Amine-containing transfection reagents and methods for making and using same

ABSTRACT

There are provided for herein novel amine-containing transfection compounds and methods for making and using same. The compounds are generally obtained by reacting a primary amine with an unsaturated compound. Transfection complexes made using the amine-containing transfection compounds in combination with additional compounds to encapsulate biologically active agents such as nucleic acids are also provided for herein. Methods of using the transfection complexes for the in vivo or in vitro delivery of biologically active agents are also described. The transfection complexes of the present invention are highly potent, thereby allowing effective modulation of a biological activity at relatively low doses compared to analogous transfection compounds known in the art.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims the right of priority under 35 U.S.C. §119 to U.S.Provisional Application Ser. No. 61/543,242, filed Oct. 4, 2011, and toU.S. Provisional Application Ser. No. 61/438,903, filed Feb. 2, 2011,and to U.S. Provisional Application Ser. No. 61/437,503, filed Jan. 28,2011, and to U.S. Provisional Application Ser. No. 61/413,905, filedNov. 15, 2210. The aforementioned applications are commonly owned withthe present application and the entire contents thereof are herebyexpressly incorporated by reference in their entirety as though fullyset forth herein

FIELD OF THE INVENTION

This invention relates generally to the field of transfection reagentsfor the in vitro and in vivo delivery of biologically active agents.More specifically, this invention relates to biodegradable andbiocompatible lipids, and transfection complexes made using same, thatmay be used to introduce nucleic acids or other biologically activeagents into cells in vitro or in vivo.

BACKGROUND

Gene therapy, such as the treatment of diseases through the applicationof nucleotide based drugs has become an important medical field.Typically, modified viruses as gene transfer vectors have been used inrecent years. However, concerns over possible undesirable side effects,such as unsolicited immune responses, when viral vectors are used haveresulted in efforts to develop non-viral alternatives (e.g., polymericdelivery systems, liposomal formulations and “naked” DNA injections).While these alternative approaches have not yet achieved the clinicaleffectiveness of viral vectors, the potential safety, processing, andeconomic benefits offered by these methods are promising.

Accordingly, better non-toxic, biodegradable, biocompatible lipids areneeded that is easily and economically efficiently prepared to be usedto transfect nucleic acids. Such lipids would have several uses,including the delivery of nucleic acids in gene therapy as well as inthe packaging and/or delivery of diagnostic, therapeutic, andprophylactic agents. The instant specification describes such newtransfection reagents and methods for synthesizing thereof.

SUMMARY

The present invention is directed towards amine-containing transfectionreagents and methods for synthesizing the same. Additional embodimentsof the present invention relate to the use of the amine-containingtransfection reagents to make transfection complexes suitable for use inthe intracellular delivery of one or more biologically active agents toa cell in vitro or a tissue in a human or an animal in vivo.

According to some embodiments of the invention, amine-containingtransfection compounds having the general structure I, orpharmaceutically acceptable salts or derivatives thereof are provided:

wherein each of X₁ and X₂ is a moiety independently selected from thegroup consisting of O, S, N-A and C-A, wherein A is selected from thegroup consisting of hydrogen and a C₁-C₂₀ hydrocarbon chain; each of Yand Z is a moiety independently selected from the group consisting ofCH—OH, C═O, C═S, S═O and SO₂; each of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is amoiety independently selected from the group consisting of hydrogen, acyclic or an acyclic, substituted or unsubstituted, branched orunbranched aliphatic group, a cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic group, asubstituted or unsubstituted, branched or unbranched acyl group, asubstituted or unsubstituted, branched or unbranched aryl group, asubstituted or unsubstituted, branched or unbranched heteroaryl group, xis an integer independently having the value between 1 and 10,inclusively, n is an integer independently having the value between 1and 3, inclusively, m is an integer independently having the valuebetween 0 and 20, inclusively, p is an integer independently having thevalue of 0 or 1, wherein if m=p=0, then R₂ is hydrogen, with the furtherproviso that if at least one of n or m has the value of 2, then R₃ andnitrogen in structure I form a moiety selected from the group consistingof:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, andeach * indicates the nitrogen atom in structure I.

According to other embodiments of the invention, there are providedamine-containing transfection compounds having the general structure IIor pharmaceutically acceptable salts thereof:

wherein when n=p=0, R₂ is H.

According to other embodiments of the invention, there are providedmethods for synthesizing amine-containing transfection compounds havingthe structure I, or pharmaceutically acceptable salts thereof, themethod comprising reacting one or more equivalents of an unsaturatedcomponent comprising at least two compounds selected from the groupconsisting of the first intermediate having the structureR₁—X₁—Y—(CR₄R₅)_(n)—Br and the second intermediate having the structureR₂—X₂—Z—(CR₆R₇)_(m)—Br, wherein in (CR₄R₅)_(n) and (CR₆R₇)_(m) portionsof the structures, each R₄ is the same or different, each R₅ is the sameor different, each R₆ is the same or different, and each R₇ is the sameor different, wherein the first and the second intermediates are thesame or different, with one equivalent of an amino component comprisinga primary amine NH₂—R₃, a diamine, a polyamine or a combination thereof.

The amine-containing transfection compounds of the present invention mayexist in neutral form or as a cation. In some embodiments, at a pH at ornear physiologically neutral (e.g. pH from about 5 to about 8), thepredominant form of an amine-containing transfection compound accordingto the presently described embodiments is a cation. In otherembodiments, a pH at or near physiologically neutral (e.g., pH fromabout 5 to about 8), the predominant form of the amine-containingtransfection compound according to the presently described embodimentsis neutral.

In a further set of non-limiting embodiments, transfection complexessuitable for the delivery of one or more biologically active agents to acell or a tissue in vitro or in vivo are provided for herein. Thetransfection complexes may include one or more of the amine-containingtransfection compounds described herein. In some embodiments, thetransfection complexes may optionally be made in combination with one ormore helper lipids, optionally in combination with one or more pegylatedlipids, optionally in combination with one or more cationic lipids, andoptionally in combination with one or more targeting moieties. In someembodiments, transfection may be made with peptide or non-peptidetransfection enhancers, fusogenic peptide or non-peptide agents, peptideor non-peptide endosomal release agents, or nuclear targeting agents(such as, e.g., a peptide containing one or more nuclear localizationsequences, such as will be readily apparent to one skilled in the artwithout undue experimentation.

Helper lipids suitable for use in the preparation and formation oftransfection complexes disclosed herein may include, though are notlimited to a cholesterol, a cholesterol derivative, one or more sterols,including phytosterols, zoosterols and hopanoids, or any of the neutralor cationic lipids that are known to allow or to facilitate theintroduction of exogenous bioactive molecules to the interior of a cellor of a tissue. In some embodiments, more than one helper lipid may beused in the formulation of the transfection complexes described herein.

Illustrative though non-limiting neutral or cationic lipids suitable foruse as helper lipids in accordance with the embodiments set forth hereinmay include saturated and unsaturated alkyl and alicyclic ethers andesters of amines, amides or derivatives thereof. Straight-chain andbranched alkyl and alkene groups of cationic lipids can contain from 1to about 25 carbon atoms. In some embodiments, straight-chain orbranched alkyl or alkene groups have six or more carbon atoms. In someembodiments, straight-chain or branched alkyl or alkene groups haveeight to about twenty carbon atoms. Alicyclic groups can contain fromabout 6 to 30 carbon atoms, or, in some embodiments eight to twentycarbon atoms. In some embodiments, the alicyclic groups includecholesterol and other steroid groups. Cationic lipids can be preparedwith a variety of counter ions (anions) including among others: Cl—,Br—, I—, F—, acetate, trifluoroacetate, sulfate, nitrite, triflate, andnitrate

Exemplary though non-limiting neutral or cationic lipids contemplatedfor use in the preparation of the presently disclosed transfectioncomplexes may include one or lipids selected from the following: BMOP(N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimunbromide), DDPES (Dipalmitoylphosphatidylethanolamine5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation ofcetyltrimethylammonium bromide (CATB) and DOPE), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE(dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine),DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC(Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine),DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC(dioleoydimethylammonium chloride), DOSPER(1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA(N-[1-(2,3-dioleyloxyl)propyl]-n,n,n-trimethylammoniumchloride), DDAB(didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate),DOTAP.Cl, DC-chol(3,β-N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA(2-(sperminecarboxamido)ethyl)-N,N-dimethy-lammonium trifluoroacetate),DC-6-14 (O,O′-Ditetradecanoyl-N-(alphatrimethylammonioacetyl)diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE(dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP(1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA(2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminiumtrifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE(N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME(N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammoniumiodide), GAP-DLRIE:DOPE(N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide/dioleyl phosphatidylethanolamine), DPPC(Dipalmitoylphosphatidylcholine), DOPG(1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl),N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivativesthereof); phosphotidylethanolamine (and derivatives thereof);phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPEdipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, O-Chol (3beta[1-ornithinamidecarbamoyl]cholesterol), POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; piperazine-based cationic lipids,phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG(dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;phosphatidylserine (and derivatives thereof); phosphatidylserines, suchas dioleoyl- or dipalmitoylphosphatidylserine; diquaternary ammoniumsalts such as N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,2-ethanediamine(TmedEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,3-propanediamine(PropEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,6-hexanediamine(HexEce), and their corresponding N,N′-dicetyl saturated analogues(TmedAce, PropAce and HexAce), diphosphatidylglycerols; fatty acidesters; monocationic transfection lipids such as1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol;1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol;1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol;1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βOH-sterolsas well as derivatives thereof phosphatidyl choline or commerciallyavailable cationic lipid mixtures such as, for example, LIPOFECTIN®CELLFECTIN® (1:1.5 (M/M) formulation ofN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS)and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXN™,TRANSFAST™, TRANSFECTAM®, TRANSMESSENGER®, vectamidine(3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine (a.k.a.diC14-amidine), OLIGOFECTAMINE®, among others. Also contemplated are anymixtures of combination of the above listed helper lipids.

Pegylated lipids suitable for use in the preparation and formation oftransfection complexes disclosed herein can be any lipid or mixture oflipids that are compatible with the formation of transfection complexesdescribed herein, and with the administration thereof to an animal or toa human in vivo, or to tissues or cells in vitro. The pegylated lipidsused in the present invention include a PEG polymer having a molecularweight between about 250 daltons and about 12,000, or in someembodiments, about 350 daltons and about 6,000 daltons, or, in someembodiments, between about 500 daltons and about 1,000 daltons, or, insome embodiments, between about 1,000 daltons and about 2,000 daltons,or, in some embodiments, between about 2,000 daltons and 5,000 daltons.

In some embodiments, the transfection complexes may include one or morebiologically active agents to be delivered to a cell or to a targettissue in vitro or in vivo. Suitable biologically active agents mayinclude any molecule that is capable of forming a transfection complexwith the presently described amine-containing transfection reagents andthat elicits a biological response when delivered to the interior of acell or cells or to a tissue in vivo or in vitro. Biologically activeagents contemplated for use in the presently described embodiments maybe cationic, neutral or anionic agents. By way of non-limiting example,exemplary biologically active agents suitable for formulation in atransfection complex may include, though are not limited to; nucleicacids (including but not limited to single or double stranded linear orcircular DNA molecules including cDNA molecules, single or doublestranded RNA molecules, small interfering RNA (siRNA) molecules, smallhairpin RNA (shRNA) molecules, microRNA (miRNA) molecules,oligonucleotides, anti-sense oligonucleotides, sense oligonucleotides),polypeptides, antibodies, oligopeptides, therapeutic peptides or proteinmolecules, peptide nucleic acids (PNAs), cationic, anionic or neutralorganic molecules or drugs, in addition to pharmaceutically acceptablesalts thereof.

In certain non-limiting illustrative embodiments of the invention,transfection complexes and methods are provided that use the compoundsof the present invention to deliver nucleic acid molecules into cells ortissues in vitro or in vivo, including the delivery of RNA interferencemolecules (RNAi) or small interfering RNA molecules (siRNA, shRNA ormiRNA) into cells for inhibition of gene expression.

In certain non-limiting illustrative embodiments, transfection complexesand methods are provided that use the compounds of the present inventionto deliver mRNA molecules into a cell or a tissue in vivo or in vitro topromote the expression of a specific protein or proteins are alsoprovided.

In other non-limiting illustrative embodiments of the invention,transfection complexes and methods are provided that use the compoundsof the present invention to deliver DNA molecules (including cDNAmolecules) into a cell or a tissue in vivo or in vitro to promote theexpression of a specific protein or proteins or to synthesize specificRNA molecules, including but not limited to mRNA molecules or RNAi ormiRNA or shRNA molecules are also provided.

In some embodiments, the transfection complexes described herein mayoptionally include one or more fusogenic or cell-penetrating peptides. Afusogenic or cell-penetrating peptide is any peptide molecule that iscapable of promoting the fusion of a lipid-containing complex to a cellmembrane (either a plasma membrane or an endosomal membrane). A varietyof fusogenic or cell-penetrating peptides are known in the art and it iswell within the skill level of a practitioner to identify suitablefusogenic or cell-penetrating peptides and condition for the use thereofin the present invention without undue experimentation.

In some embodiments, the transfection complexes described herein mayoptionally include one or more transfection helpers or targetingmoieties. A targeting moiety may be a peptide, a modified peptide, anantibody, a modified antibody, a receptor molecule, a modified receptormolecule, a single or a double stranded nucleic acid molecule, amodified single or double stranded nucleic acid molecule, a peptide ornucleic acid aptamer, a modified peptide or nucleic acid aptamer, anorganic molecule, a polysaccharide, or any other molecule that iscapable of targeting a transfection complex to specific tissue or celltype for targeted delivery of a biologically agent thereto, such as willbe readily apparent to have having ordinary skill level in the art. Insome embodiments, modification of a peptide, an antibody, a nucleicacid, an aptamer, and the like may include conjugating the peptide,antibody, nucleic acid, aptamer, and the like to a PEG moiety.Alternatively, modification of a peptide, an antibody, a nucleic acid,an aptamer, and the like may include conjugating the peptide, antibody,nucleic acid, aptamer, and the like to a PEG-lipid moiety A variety oftargeting moieties are widely known to those skilled in the art, and allare contemplated for use with the presently described embodiments,without limitation.

In some embodiments, the transfection complexes provided for herein maybe stable for up to 1 year and may either be contacted with the cells ortissues to be transfected, or be administered to an animal or to a humanimmediately or shortly after being formed, or optionally may stored fora period of time prior to being contacted with the cells or tissues, orbeing administered to an animal or a human. The transfection complexesare stable and may be stored for a time period of at least 30 minutes,at least 45 minutes, at least 1 hour, at least 2 hours, at least 3hours, at least 4 hours, at least 5 hours, at least 10 hours, at least15 hours, at least 20 hours, at least 24 hours, at least 48 hours, atleast 72 hours, at least 5 days, at least 7 days, at least 14 days, atleast 28 days, at least 1 month, at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months or at least 1year at room temperature, or at a temperature greater than freezing, upto about room temperature. It is to be understood that the formulationdescribed herein may include one or more stabilizing agents,preservatives, buffers, etc, that aid in the long-term stabilization andstorage of bioactive formulation, such as will be readily understood bythe skilled practitioner of the biological and pharmaceutical arts, andwithout requiring undue experimentation to achieve. It is alsounderstood, that the storage period can be between any of these timeperiods, for example between 31 minutes and 1 hour or between 1 hour and24 hours.

In some embodiments, methods for the preparation of functionaltransfection complexes are provided. The methods generally includeforming a lipid-aggregate by encapsulating a biologically active agentin a composition containing one or more of the amine-containingtransfection compounds described herein, optionally in combination withone or more helper lipids, stabilizing lipids, transfection helpers,pegylated lipids or targeting moieties. Such methods may include a1)mixing one or more amine-containing transfection compounds, at least onehelper lipid, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, in an alcohol/aqueous solutionwherein the alcohol concentration is <50%; a2) mixing one or moreamine-containing transfection compounds, at least one helper, optionallymore than one helper lipid and one or more pegylated lipids, or a saltthereof, in a molar percentage such that the one or moreamine-containing transfection compounds are present at 15%-50%; a3)mixing one or more amine-containing transfection compounds, at least onehelper lipid, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, in a molar percentage such that thePegylated lipids are present at <50%; and a4) mixing one or moreamine-containing transfection compounds, at least one helper lipid,optionally more than one helper lipid and one or more pegylated lipids,or a salt thereof, wherein the pegylated lipid has a polyethylene glycolmolecular weight of about 2000-12000 and a fatty acid chain length ofC₆-C₂₀ alkyl, or C₁₀-C₂₀ alkenyl; and complexing the lipid aggregate inan alcohol/aqueous solution with the bioactive agent to form atransfection complex, wherein the alcohol concentration is <50%,preferably less than 40%. In some embodiments, the method includes a1)and a2), a2) and a3), a1) and a3), a2) and a4), a3) and a4), a1) anda4), or a1)-a4), for example. In some embodiments, the alcohol is aC1-C4 alcohol. In some embodiments, the alcohol is ethanol. In someembodiments, the alcohol is a pharmaceutically acceptable alcohol suchas an alcohol that is liquid at about room temperature, for example,ethanol, propylene glycol, 2-(2-ethoxyethoxyl)ethanol (Transcutol™),benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol300, polyethylene glycol 400 or a mixture thereof. In some embodiments,the alcohol for mixing is different than the alcohol for complexing.

Further embodiments described herein provide for methods to screen largenumbers of transfection compounds for tissue-biased delivery in vivo.Such methods may include preparing a plurality of transfection complexescontaining a compound that readily facilitates the detection of a markerin combination with a test transfection compound, delivering each of theplurality of transfection complexes to a test animal, and detecting themarker.

In some embodiments, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of transfectioncomplexes, each transfection complex having at least one testtransfection compound in combination with at least one nucleic acid thatfacilitates detection of delivery to a tissue. The nucleic acid may bean RNA molecule or a DNA molecule that encodes a protein that can bedirectly detected (such as, e.g., Green Fluorescent Protein (GFP), redFluorescent Protein, Luciferase, or the like), or encode a protein thateffects expression of a protein that can be directly detected.

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes Green Fluorescent Protein. Each unique transfection complex maybe delivered either intravenously, subcutaneously, or to a tissue to atest animal, such as a mouse. After a predetermined amount of time,tissues from the mouse may be harvested and the expression of GFP invarious tissues may be detected by gross examination, histologicalexamination or by molecular detection (PCR, Western blotting, or thelike) to determine which to tissue or tissues transfection complexescontaining specific transfection compounds are delivered to.

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes Luciferase. Each unique transfection complex may be deliveredeither intravenously, subcutaneously, or to a tissue to a test animal,such as a mouse. After a predetermined amount of time, tissues from themouse may be harvested and the expression of Luciferase in varioustissues may be detected by gross examination, histological examinationor by molecular detection (PCR, Western blotting, or the like), orimaged in-vivo using the IVIS® Imaging System (Caliper).

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes a specific transcription factor. Each unique transfectioncomplex may be delivered either intravenously, subcutaneously, or to atissue to a transgenic animal that expresses a reporter gene (such as,e.g., luciferase) under the control of the specific transcriptionfactor. After a predetermined amount of time, tissues from thetransgenic animal may be harvested and the expression of reporter genein various tissues may be detected by gross examination, histologicalexamination or by molecular detection (PCR, Western blotting, or thelike). If the reporter gene is luciferase, detection may be accomplishedin-vivo using the IVIS® Imaging System (Caliper).

These and other features of the present invention will become betterunderstood with reference to the following description, drawings, andappended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a graph depicting some properties of a lipid compositionprepared using compounds according to some embodiments of the presentinvention;

FIG. 2 shows a graph depicting some properties of a lipid compositionprepared using compounds according to other embodiments of the presentinvention;

FIG. 3 shows a graph depicting some properties of a lipid compositionprepared using compounds according to other embodiments of the presentinvention;

FIG. 4 shows a graph depicting some properties of a lipid compositionprepared using compounds according to other embodiments of the presentinvention; and

FIG. 5 shows a graph depicting some properties of a lipid compositionprepared using compounds according to other embodiments of the presentinvention.

FIGS. 6 A and 6B shows whole animal and whole tissue mount imagesdepicting some properties of a lipid composition prepared usingcompounds according to other embodiments of the present invention.

FIG. 7A-7F show graphs depicting some properties of a lipid compositionprepared using compounds according to other embodiments of the presentinvention.

DETAILED DESCRIPTION

Reference will now be made in detail to some embodiments of theinvention. While the invention will be described in conjunction with theembodiments discussed below, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover alternatives, modifications, andequivalents, which is included within the invention as defined by theappended claims.

I. DEFINITIONS AND ABBREVIATIONS

It is to be understood that the present invention is not limited toparticular devices or biological systems, which may, of course, vary. Itis also to be understood that, as used in this specification and theappended claims, the singular forms “a”, “an”, and “the” includesingular and plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a lipid” includes one ormore lipids. It is to be yet further understood that any terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

The terms used throughout this specification generally have theirordinary meanings in the art, within the context of the invention, andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the generalembodiments of the invention, as well as how to make and use them. Itwill be readily appreciated that the same thing can be said in more thanone way. Consequently, alternative language and synonyms may be used forany one or more of the terms discussed herein, nor is any specialsignificance to be placed upon whether or not a term is elaborated ordiscussed in greater detail herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofother synonyms. The use of examples anywhere in this specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term.

Compounds of the present invention may exist in particular geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and toms-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof, as falling within the scope of theinvention. Additional asymmetric carbon atoms is present in asubstituent such as an alkyl group. All such isomers, as well asmixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios isutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures. If, for instance, a particularenantiomer of a compound of the present invention is desired, it isprepared by asymmetric synthesis, or by derivation with a chiralauxiliary, where the resulting diastereomeric mixture is separated andthe auxiliary group cleaved to provide the pure desired enantiomers.Alternatively, where the molecule contains a basic functional group,such as amino, or an acidic functional group, such as carboxyl,diastereomeric salts are formed with an appropriate optically-activeacid or base, followed by resolution of the diastereomers thus formed byfractional crystallization or chromatographic means well known in theart, and subsequent recovery of the pure enantiomers.

Unless stated otherwise, the following terms, definitions, andabbreviations as used herein are intended to have the followingmeanings:

The term “protecting group,” as used herein, refers to a group thattemporarily blocks a particular functional moiety, e.g., O, S, or N, isso that a reaction is carried out selectively at another reactive sitein a multifunctional compound. A protecting group reacts selectively ingood yield to give a protected substrate that is stable to the projectedreactions, and the protecting group is selectively removable in goodyield by readily available reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative; and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction.

As detailed herein, oxygen, sulfur, nitrogen, and carbon protectinggroups is utilized. Non-limiting examples of exemplary hydroxylprotecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl(MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl,4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM),2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl,4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,4-methoxytetrahydrothiopyranyl S,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxyl)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl iV-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl,di(p-niethoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxy acetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

For protecting 1,2- or 1,3-diols, non-limiting examples of exemplaryprotecting groups include methylene acetal, ethylidene acetal,1-t-butylethylidene ketal, 1-phenylethylidene ketal,(4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal,acetonide, cyclopentylidene ketal, cyclohexylidene ketal,cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal,2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethyleneacetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester,1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester,a-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidenederivative, a-(N,N′-dimethylamino)benzylidene derivative,2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

Non-limiting examples of exemplary amino-protecting groups includemethyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate, 2,7-di-f-butyl-[9-(10,10-dioxo-10, 10, 10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc),2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ),1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocimiamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, NN′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMB S),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

The term “substituted” whether used alone or is preceded by the term“optionally,” and substituents contained in formulae of this invention,refer to the replacement of hydrogen radicals in a given structure withthe radical of a specified substituent. When more than one position inany given structure is substituted with more than one substituentselected from a specified group, the substituent is either the same ordifferent at every position. The term “substituted” is inclusive of allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Heteroatoms may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valencies of the heteroatoms.Furthermore, this invention is not intended to be limited in any mannerby the permissible substituents of organic compounds. Combinations ofsubstituents and variables are those that result in the formation ofstable compounds.

The term “stable,” as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. The term “aliphatic” isinclusive of, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

The term “alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl” or“alkynyl.” The terms “alkyl,” “alkenyl” and “alkynyl” encompass bothsubstituted and unsubstituted groups. The alkyl, alkenyl, and alkynylgroups employed in the invention contain 1-20 aliphatic carbon atoms.“Lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched or unbranched) having 1-6 carbonatoms.

Exemplary aliphatic groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂-cyclopropyl, vinyl, allyl,n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH₂-cyclobutyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,—CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, and —CH₂-cyclohexylmoieties which is one or more substituents. Exemplary alkenyl groupsinclude, but are not limited to, ethenyl, propenyl, butenyl and1-methyl-2-buten-1-yl. Representative alkynyl groups include, but arenot limited to, ethynyl, 2-propynyl (propargyl) and 1-propynyl.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. Exemplary alkyl radicals include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

The term “alkenyl,” as used herein, refers to a monovalent group derivedfrom a hydrocarbon moiety having at least one carbon-carbon double bondby the removal of a single hydrogen atom. Exemplary alkenyl groupsinclude, but are not limited to, ethenyl, propenyl, butenyl and1-methyl-2-buten-1-yl.

The term “alkynyl,” as used herein refers to a monovalent group derivedfrom a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Exemplary alkynyl groups include, butare not limited to, ethynyl, 2-propynyl (propargyl) and 1-propynyl, andthe like.

The terms “alkoxy” and “thioalkyl,” as used herein refer to an alkylgroup, as previously defined, attached to the parent molecule through anoxygen atom or through a sulfur atom, respectively. In certainembodiments, the alkyl, alkenyl, and alkynyl groups contain 1-20aliphatic carbon atoms. Exemplary alkoxy groups, include but are notlimited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy,neopentoxy and n-hexoxy. Exemplary thioalkyl groups include, but are notlimited to, methylthio, ethylthio, propylthio, isopropylthio andn-butylthio.

The term “alkylamino,” as used herein, refers to a group having thestructure —NHR′, wherein R′ is aliphatic, as defined above, containing1-20 aliphatic carbon atoms. Exemplary alkylamino groups include, butare not limited to, methylamino, ethylamino, n-propylamino,iso-propylamino, cyclopropylamino, n-butylamino, tert-butylamino,neopentylamino, n-pentylamino, hexylamino and cyclohexylamino.

The term “dialkylamino,” as used herein, refers to a group having thestructure —NRR₁, wherein R and R₁ are each an aliphatic group, asdefined herein, containing 1-20 aliphatic carbon atoms. R and R₁ is thesame or different or is linked to form an aromatic or non-aromaticcyclic structure. Exemplary dialkylamino groups include, but are notlimited to, dimethylamino, methyl ethylamino, diethylamino,methylpropylamino, di(n-propyl)amino, di(iso-propyi)amino,di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino,di(neopentyl)amino5 di(n-pentyl)amino, di(hexyl)amino anddi(cyclohexyl)amino. Exemplary cyclic diaminoalkyl groups include, butare not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl,pyrrolyl, imidazolyl, 1,3,4-trianolyl and tetrazolyl.

The term “carboxylic acid,” as used herein, refers to a compoundcomprising a group of formula —COOH.

Some examples of substituents of the above-described aliphatic and othermoieties of compounds of the invention include, but are not limited toaliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl,heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy,alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I,—OH, —NO₂, —CN, —CF₃, —CH₂CF₃, —CHCl₂, —CH₂OH, —CH₂CH₂OH, —CH₂NH₂,—CH₂SO₂CH₃, —C(O)R_(x), —CO₂(R_(x)), —CON(R_(x))₂, —OC(O)R_(x),—OCO₂R_(x), —OCON(R_(x))₂, —N(R_(x))₂, —S(O)₂R_(x) and —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and herein issubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein is substituted or unsubstituted.

The terms “aryl” and “heteroaryl,” as used herein, refer to stable mono-or polycyclic, heterocyclic, polycyclic, and polyheterocyclicunsaturated moieties having 3-14 carbon atoms, each of which issubstituted or unsubstituted. Substituents include, but are not limitedto, any of the substituents recited above for aliphatic moieties. Theterm “aryl” is inclusive of mono- or bicyclic carbocyclic ring systemshaving one or two aromatic rings including, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl and The term “heteroaryl” isinclusive of cyclic aromatic radicals having from five to ten ringatoms, of which 1-3 ring atoms is selected from S, O, and N, and theremaining ring atoms are carbon, the radical being joined to the rest ofthe molecule via any of the ring atoms, such as, for example, pyridyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl or isoquinolinyl.

Aryl and heteroaryl groups is unsubstituted or substituted, whereinsubstitution includes replacement of one, two, three, or more of thehydrogen atoms thereon independently with any one or more of thefollowing moieties including, but not limited to aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy,aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio,heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO₂, —CN, —CF₃,—CH₂CF₃, —CHCl₂, —CH₂OH, —CH₂CH₂OH, —CH₂NH₂, —CH₂SO₂CH₃, —C(O)R_(x),—CO₂(R_(x)), —CON(R_(x))₂, —OC(O)R_(x), —OCO₂R_(x), —OCON(R_(x))₂,—N(R_(x))₂, —S(O)₂R_(x) and —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein is substitutedor unsubstituted, branched or unbranched, cyclic or acyclic, and whereinany of the aryl or heteroaryl substituents described above and herein issubstituted or unsubstituted.

The term “cycloalkyl,” as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl, which may optionally besubstituted with substituents including, but not limited to aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy,aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio,heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO₂, —CN, —CF₃,—CH₂CF₃, —CHCl₂, —CH₂OH, —CH₂CH₂OH, —CH₂NH₂, —CH₂SO₂CH₃, —C(O)R_(x),CO₂(R_(x)), —CON(R_(x))₂, —OC(O)R_(x), —OCO₂R_(x), —OCON(R_(x))₂,—N(R_(x))₂, —S(O)₂R_(x) and —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and is substituted orunsubstituted, branched or unbranched, cyclic or acyclic, and whereinany of the aryl or heteroaryl substituents described above and herein issubstituted or unsubstituted.

The term “heteroaliphatic,” as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesis branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc.Heteroaliphatic moieties are substituted by independent replacement ofone or more of the hydrogen atoms thereon with one or more moietiesincluding, but not limited to aliphatic, heteroaliphatic, aryl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy,heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, —F,—Cl, —Br, —I, —OH, —NO₂, —CN, —CF₃, —CH₂CF₃, —CHCl₂, —CH₂OH, —CH₂CH₂OH,—CH₂NH₂, —CH₂SO₂CH₃, —C(O)R_(x), —CO₂(R_(x)), —CON(R_(x))₂, —OC(O)R_(x),—OCO₂R_(x), —OCON(R_(x))₂, —N(R_(x))₂, —S(O)₂R_(x) and —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and herein issubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein is substituted or unsubstituted.

The terms “halo” and “halogen,” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl and trifluoromethyl.

The term “heterocycloalkyl” or “heterocycle,” as used herein, refers toa non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms isoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings is fused to abenzene ring. Representative heterocycles include, but are not limitedto, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuryl. Theterm a “substituted” heterocycloalkyl or heterocycle group is utilizedand as used herein, refers to a heterocycloalkyl or heterocycle group,as defined above, substituted by the independent replacement of one, twoor three of the hydrogen atoms thereon with but are not to aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy,aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio,heteroalkylthio, heteroarylthio, —F, —Cl, —Br, —I, —OH, —NO₂, —CN, —CF₃,—CH₂CF₃, —CHCl₂, —CH₂OH, —CH₂CH₂OH, —CH₂NH₂, —CH₂SO₂CH₃, —C(O)R_(x),—CO₂(R_(x)), —CON(R_(x))₂, —OC(O)R_(x), —OCO₂R_(x), —OCON(R_(x))₂,—N(R_(x))₂, —S(O)₂R_(x) and —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein is substitutedor unsubstituted, branched or unbranched, cyclic or acyclic, and whereinany of the aryl or heteroaryl substituents described above and herein issubstituted or unsubstituted.

Exemplary non-limiting heterocyclic and aromatic heterocyclic groupsthat is included in the compounds of the invention include3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,A-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,A-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl) piperazine, 4-(2-nitrophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,A-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl) piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine,4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term “carbocycle,” as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is a carbon atom.

The term “independently selected” is used herein to indicate that thegroups is identical or different.

As used herein, the term “labeled” is intended to mean that a compoundhas at least one element, isotope, or chemical compound attached toenable the detection of the compound by using a radioactive or heavyisotope label, or an immune label such as an antibody or antigen or alabel derived from a colored, luminescent, phosphorescent, orfluorescent dye. Photoaffinity labeling employing, for example, o-, m-and p-azidobenzoyls, substituted with one or more halogen moieties,including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid,is utilized for the direct elucidation of intermolecular interactions inbiological systems.

The term “animal,” as used herein, may refer to humans as well asnon-human animals, including, for example, mammals (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig),birds, reptiles, amphibians, and fish.

The term “cell” generally refers to eukaryotic cells of any type andfrom any source. Types of eukaryotic cells include epithelial,fibroblastic, neuronal, hematopoietic cells and the like from primarycells, tumor cells or immortalized cell lines. Sources of such cellsinclude any animal such as human, canine, mouse, hamster, cat, bovine,porcine, monkey, ape, sheep, fish, insect, fungus, and any plantincluding crop plants, algae, ornamentals and trees.

“Delivery” is used to denote a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on, the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell and delivery vialipid aggregates or transfection complexes a means for delivering thedesired compound to the appropriate cellular compartment within a cell.In certain uses, especially under in vivo conditions, delivery to aspecific target cell type is preferable and can be facilitated bycompounds of the invention.

Drug refers to any therapeutic or prophylactic agent other than foodwhich is used in the prevention, diagnosis, alleviation, treatment, orcure of disease in man or animal.

“Kit” refers to transfection or protein expression kits which includeone or more of the compounds of the present invention or mixturesthereof. Such kits may comprise a carrying means being compartmentalizedto receive in close confinement one or more container means such asvials, test tubes and the like. Each of such container means comprisescomponents or a mixture of components needed to perform transfection.Such kits may include one or more components selected from nucleic acids(preferably one or more vectors), cells, one or more compounds of thepresent invention, lipid-aggregate forming compounds, transfectionenhancers, biologically active substances, etc.

The term “associated with”, when used in the context of molecularinteractions, refers to two entities linked by a direct or indirectcovalent or non-covalent interaction, such as hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

The term “biocompatible,” as used herein refers to compounds that arenot toxic to cells. Compounds are biocompatible if their addition tocells in vitro results in less than or equal to 20% cell death, andtheir administration in vivo does not induce inflammation or other suchadverse effects.

The term “biodegradable,” as used herein, refers to compounds that, whenintroduced into cells, are broken down into components that the cellscan either reuse or dispose of without significant toxic effect on thecells (i.e., fewer than about 20% of the cells are killed when thecomponents are added to cells in vitro). The components do not induceinflammation or other adverse effects in vivo. The chemical reactionsrelied upon to break down the biodegradable compounds are typicallyuncatalyzed. The term “effective amount,” as used herein with respect toan active agent, refers to the amount necessary to elicit the desiredbiological response. The effective amount of an agent or device may varydepending on such factors as the desired biological endpoint, the agentto be delivered, the composition of the encapsulating matrix, the targettissue, etc. Delivery of an “effective amount of a molecule” is thedelivery of the molecule into a cell in sufficient amounts so that themolecule elicits a biological response, for example, modulating theexpression of one or more genes in the cell. In specific embodiments, aneffective amount of a molecule is delivered to a cell such that anamelioration or improvement in a disease, condition, or disorder relatedto the cell can be obtained. Delivery of an “effective amount of siRNA”or an “effective amount or RNAi” is the delivery of siRNA or other RNAiinto a cell in sufficient amounts to cause a reduction in expression ofthe target gene in the cell.

The terms “biologically active agent”, “bioactive agents” or the like,generally refers to a composition, complex, compound or molecule whichhas a biological effect or that modifies, causes, promotes, enhances,blocks or reduces a biological effect, or that enhances or limits theproduction or activity of, reacts with and/or binds to a secondmolecules which has a biological effect. The second molecule can, butneed not be, an endogenous molecule (e.g., a molecule, such as a proteinor nucleic acid, normally present in the target cell). A biologicaleffect may be, but is not limited to, one that stimulates or causes animmunoreactive response; one that impacts a biological process in acell, tissue or organism (e.g., in an animal); one that imparts abiological process in a pathogen or parasite; one that generated orcauses to be generated a detectable signal; one that regulates theexpression of a protein or polypeptide; one that stops or inhibits theexpression of a protein or polypeptide; or one that causes or enhancesthe expression of a protein or polypeptide. Biologically activecompositions, complexes, compounds or molecules may be used ininvestigative, therapeutic, prophylactic and diagnostic methods andcompositions and generally act to cause.

The term “cationic lipid” refers to any cationic lipids which may beused for transfection and which under physiological conditions possessat least one positive charge. While it is to be understood that certainof the amine-containing transfection agents that form the basis of thepresent disclosure also exist as cations under physiological conditions,the term is also extended without limitation to any cationic helperlipids that may be used to co-formulate transfection complexes asdescribed herein. Additional cationic lipids other than the novelamine-containing transfection agents described herein may include, butnot limited to, e.g., DOSPA, DOTMA, DMRIE, DOT AP, DOGS and TMTPS, aswell as any of the cationic lipids described herein as “helper lipid”.

“Target cell” or “target tissue” refers to any cell or tissue to which adesired compound is delivered, using a lipid aggregate or transfectioncomplex as carrier for the desired compound.

Transfection is used herein to mean the delivery of any nucleic acid,protein or other macromolecule to a target cell or tissue in vitro or invivo (i.e., in an animal, a plant or a human), such that the nucleicacid, protein or other macromolecule is expressed in, confers aphenotype to, or has a biological function in the cell.

The term “expressible nucleic acid” includes both DNA and RNA withoutregard to molecular weight, and the term “expression” means anymanifestation of the functional presence of the nucleic acid within thecell including, without limitation, both transient expression and stableexpression.

The term “transfection complex”, as used herein generally refers to acomposition formulated for the delivery of a biologically active agent,such as a nucleic acid, a protein, a macromolecule, or the like, to acell or to a tissue in vivo or in vitro. Transfection complexes as usedherein may include at least one or more of the amine-containingtransfection compounds in combination with the biologically activecompound to be delivered, optionally in combination with one or morehelper lipids, one or more pegylated lipids, one or more targetingmoieties, in addition to the bioactive agent that is to be delivered.For the purposes described herein, the term “transfection complex” maybe thought of as a lipoplex or a lipid aggregate contacted with abioactive agent. Thus, in some instances in the following disclosure,terms such as lipoplex, lipid aggregate and the like may be used to makereference a transfection complex that lacks the one or more bioactiveagents or “payloads”.

The term “helper lipid”, as used herein, generally refers to a lipidthat is suitable for use in the preparation and formation oftransfection complexes disclosed herein. Suitable helper lipids mayinclude, though are not limited to cholesterols, cholesterolderivatives, sterols, including phytosterols, zoosterols and hopanoids,or any of the neutral or cationic lipids that are known to allow or tofacilitate the introduction of exogenous bioactive molecules to theinterior of a cell or of a tissue. In some embodiments, more than onehelper lipid may be used in the formulation of the transfectioncomplexes described herein. Exemplary though non-limiting neutral orcationic lipids contemplated for use in the preparation of the presentlydisclosed transfection complexes may include one or lipids selected fromthe following: BMOP(N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimunbromide), DDPES (Dipalmitoylphosphatidylethanolamine5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation ofcetyltrimethylammonium bromide (CATB) and DOPE), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE(dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine),DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC(Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine),DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC(dioleoydimethylammonium chloride), DOSPER(1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA(N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride), DDAB(didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate),DOTAP.Cl, DC-chol(3,β-N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA(2-(sperminecarboxamido)ethyl)-N,N-dimethy-lammonium trifluoroacetate),DC-6-14 (O,O′-Ditetradecanoyl-N-(alphatrimethylammonioacetyl)diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE(dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP(1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA(2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminiumtrifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE(N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME(N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammoniumiodide), GAP-DLRIE:DOPE(N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide/dioleyl phosphatidylethanolamine), DPPC(Dipalmitoylphosphatidylcholine), DOPG(1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl),N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivativesthereof); phosphotidylethanolamine (and derivatives thereof);phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPEdipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, O-Choi (3beta[1-ornithinamidecarbamoyl]cholesterol), POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; piperazine-based cationic lipids,phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG(dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;phosphatidylserine (and derivatives thereof); phosphatidylserines, suchas dioleoyl- or dipalmitoylphosphatidylserine; diquaternary ammoniumsalts such as N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,2-ethanediamine(TmedEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,3-propanediamine(PropEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,6-hexanediamine(HexEce), and their corresponding N,N′-dicetyl saturated analogues(TmedAce, PropAce and HexAce), diphosphatidylglycerols; fatty acidesters; monocationic transfection lipids such as1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol;1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol;1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol;1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βOH-sterolsas well as derivatives thereof phosphatidyl choline or commerciallyavailable cationic lipid mixtures such as, for example, LIPOFECTIN®CELLFECTIN® (1:1.5 (M/M) formulation of N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS) anddioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXN™,TRANSFAST™, TRANSFECTAM®, TRANSMESSENGER®, vectamidine(3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine (a.k.a.diC14-amidine), OLIGOFECTAMINE®, among others. Also contemplated are anymixtures of combination of the above listed helper lipids. The followingpatent documents, patent applications, or references are incorporated byreference herein in their entirety and in particular for theirdisclosure of transfection agents containing cationic and neutral helperlipids which may be used to comprise the transfection complexes of thepresent invention: U.S. Pat. Nos. 6,075,012; 6,020,202; 5,578,475;5,736,392; 6,051,429; 6,376,248; 5,334,761; 5,316,948; 5,674,908;5,834,439; 6,110,916; 6,399,663; 6,716,882; 5,627,159;PCT/US/2004/000430, published as WO 04063342 A2; PCT/US/9926825,published as WO 0027795 A1; PCT/US/04016406, published as WO 04105697;and PCT/US2006/019356, published as WO 07130073 A2.

The term “pegylated lipid” as used herein generally refers to a lipidthat is covalently conjugated to one or more polyethylene glycolmoieties. Pegylated lipids for lipoplex embodiments herein includephosphatidylethanolamine (PE) based pegylated lipids such as, forexample,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-MW] where MW refers to average MW of the polyethylene glycolmoiety. Such dimyristoyl-PEG-PE lipids are commonly designated 14:0 PEG(MW) PE. The average MW of the polyethylene glycol moiety can be 25,350, 550, 750, 1000, 2000, 3000, 5000, 6000, 8000 or 12000, for example.The fatty acid chains of the phosphatidylethanolamine based pegylatedlipids may include, for example, a 1,2-dioleoyl group such as for 18:1PEG (MW) PE, a 1,2-dipalmitoyl group such as for 16:0 PEG (MW) PE, or a1,2-distearoyl-group such as for 18:0 PEG (MW) PE. Furtherphosphatidylethanolamine (PE) based pegylated lipids include, forexample,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[MOD(polyethyleneglycol)-MW], also referred to as DSPE-MOD PEG(MW) wherein MOD refers toa functional moiety such as an amine, biotin, carboxylic acid, folate,maleimide, PDP, or carboxyfluorescein moiety. The MW may be 2000 or5000, for example. Pegylated lipids for the embodiments described hereinalso include ceramide based pegylated lipids such as, for example,N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)MW]},designated C8 PEG (MW) ceramide, where MW is 750, 2000, or 5000, forexample. Alternatively, the fatty acid moiety may have an N-palmitoyl(C16) group such as for C16 PEG (MW) ceramide.

A “liposomal composition” generally is a formulation that includes oneor more liposomes. In some instances, the term “liposomal composition”may be used interchangeably with the term “transfection complex”. Theseformulations are typically colloids, but can be dried formulations aswell. A liposome is a vesicular colloidal particle composed of selfassembled amphiphilic molecules. Liposomal compositions of the presentinvention typically include at least one or more cationic lipids eitheralone or optionally in combination with one or more helper lipids (i.e.,a neutral lipid, a cholesterol or cholesterol derivative, a cationiclipid) that are processed using standard methods to form aliposome-containing colloid suspension. Liposomal compositions of thepresent invention are those containing one or more amine-containingtransfection lipids, one or more helper lipids, one or more pegylatedlipids, optionally, in combination with one or more neutral and/orhelper lipids or targeting moieties which are treated by any of thestandard methods known in the art without limitation to form liposomes.Liposomal compositions can be distinguished one from another by particlesize measurements. Different compositions will exhibit differences inparticle size and uniformity of particle size, e.g., average particlesize, and polydispersity. Different compositions will exhibitdifferences in the extent of the composition that is in the form ofliposomes. In some non-limiting embodiments, liposomal compositions willexhibit particle size in the range 120 nm and 800 nm and will exhibitgenerally lower polydispersity. Lipoplex particle size (with siRNA orother cargo) may range from about 40 nm to 135 nm. In some embodiments,lipoplex particle size is 50 nm to 120 nm, 50 nm to 100 nm, 60 nm to 90nm, 70 nm to 90 nm, or about 85 nm.

The term “Lipid aggregate” or “lipoplex” is a generic term that includesliposomes of all types, both unilamellar and multilamellar, as well asvesicles, micelles and more amorphous aggregates. A cationic lipidaggregate is a lipid aggregate comprising a combination of one or morecationic compounds, optionally in combination with non-cationic lipids(including neutral lipids), such that the lipid aggregate has a netpositive charge. Amine-containing transfection compounds of the presentinvention can form a lipid aggregate, optionally with a helper lipid andfurther optionally with one or more pegylated lipids and/or one or moretargeting moieties, which can then form a lipid-bioactive agent complexwhen contacted with a suitable bioactive agent. The terms “lipidaggregate” or “lipoplex” are generally used herein to refer to a “naked”transfection complex, i.e., a transfection complex that generally lacksa payload of bioactive agent to be delivered to a cell or to a tissue invitro or in vivo.

The term “lipid-bioactive agent” generally refers to the noncovalentassociation between a lipid or lipid aggregate and a bioactive agent,such as a nucleic acid, a polypeptide, and the like.

As used herein “nucleic acid” and its grammatical equivalents willinclude the full range of polymers of single or double strandednucleotides and includes nucleic acids (including DNA, RNA, and DNA-RNAhybrid molecules) that are isolated from a natural source; that areprepared in vitro, using techniques such as PCR amplification orchemical synthesis; that are prepared in vivo, e.g., via recombinant DNAtechnology; or that are prepared or obtained by any known method. Anucleic acid typically refers to a polynucleotide molecule comprised ofa linear strand of two or more nucleotides (deoxyribonucleotides and/orribonucleotides) or variants, derivatives and/or analogs thereof. Theexact size will depend on many factors, which in turn depends on theultimate conditions of use, as is well known in the art. The nucleicacids of the present invention include without limitation primers,probes, oligonucleotides, vectors, constructs, plasmids, genes,transgenes, genomic DNA, cDNA, RNA, RNAi, siRNA, shRNA, stRNA, PCRproducts, restriction fragments, oligonucleotides and the like.

As used herein, the term “nucleotide” includes any monomeric unit of DNAor RNA containing a sugar moiety (pentose), a phosphate, and anitrogenous heterocyclic base and may also include mono-, di- andtriphosphate forms of such nucleotides. The base is usually linked tothe sugar moiety via the glycosidic carbon (at the 1′ carbon of pentose)and that combination of base and sugar is called a “nucleoside.” Thebase characterizes the nucleotide with the four customary bases of DNAbeing adenine (A), guanine (G), cytosine (C) and thymine (T). Inosine(I) is an example of a synthetic base that can be used to substitute forany of the four, naturally occurring bases (A, C, G, or T). The four RNAbases are A, G, C, and uracil (U). Accordingly, a nucleic acid may be anucleotide sequence comprising a linear array of nucleotides connectedby phosphodiester bonds between the 3′ and 5′ carbons of adjacentpentoses. Other modified nucleotides are known and may be sued in thepractice of the invention. The term nucleotide includes ribonucleosidetriphosphates ATP, UTP, ITP, CTG, GTP and deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTPand 7-deaza-dATP, and nucleotide derivatives that confer nucleaseresistance on the nucleic acid molecule containing them. The termnucleotide as used herein also refers to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrated examples ofdideoxyribonucleoside triphosphates include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the presentinvention, a “nucleotide” may be unlabeled or detectably labeled by wellknown techniques. Detectable labels include, for example, radioactiveisotopes, fluorescent labels, chemiluminescent labels, bioluminescentlabels and enzyme labels. Various labeling methods known in the art canbe employed in the practice of this invention.

“RNA” or “RNA molecule” refers to any RNA molecule or functional portionthereof, of any size and having any sequence, from any source, includingRNA from viral, prokaryotic, and eukaryotic organisms. The RNA moleculemay be chemically modified and in any form, including, but not limitedto, linear or circular, and single or double stranded. Non-limitingexamples of RNA molecules include rRNA, tRNA, mRNA, mtRNA, tmRNA, RNAi,siRNA, shRNA, and stRNA. In some embodiments, siRNA molecules useful inthe practice of the invention include, for example, those described inU.S. patent application Ser. No. 10/357,529 published as U.S.2004/0014956, Ser. No. 10/357,826 published as U.S. 2004/0054155, Ser.No. 11/049,636 published as U.S. 2006/0009409, Ser. No. 11/776,313published as U.S. 2009/0023216, and Ser. No. 12/062,380 filed Apr. 3,2008; and as described in PCT published applications PCT/US03/03223published as WO 2003/064626, and PCT/US03/03208 published as WO03/064625, which U.S. and PCT applications are incorporated by referenceherein. Further siRNA molecules useful in the practice of the inventioninclude, for example, those described in PCT patent applicationPCT/US2008/076675 published as WO 2009/039173 on Mar. 26, 2009; whichapplication is incorporated by reference herein.

The terms “peptide”, “polypeptide”, or “protein,” as used herein referto a string of at least three amino acids linked together by peptidebonds. The terms “protein” and “peptide” may be used interchangeably,though it is generally understood that a “polypeptide” or “protein” islarger than a peptide. “Peptide” may refer to an individual peptide or acollection of peptides.

The terms “polynucleotide” or “oligonucleotide,” as used herein, referto a polymer of nucleotides. Typically, a polynucleotide comprises atleast three nucleotides. The polymer may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term “lipid” refers to hydrophobic or amphiphilic organic compoundsinclusive of fats, oils and triglyderides.

II. EMBODIMENTS OF THE INVENTION Amine-Containing Transfection Compounds

The present invention describes various amine-containing compoundsuseful as transfection reagents and methods of synthesizing thereof.More particularly, according to some embodiments of the invention, thereare provided compounds having the general structure I, orpharmaceutically acceptable salts thereof:

wherein each of X₁ and X₂ is a moiety independently selected from thegroup consisting of O, S, N-A and C-A, wherein A is selected from thegroup consisting of hydrogen and a C₁-C₂₀ hydrocarbon chain; each of Yand Z is a moiety independently selected from the group consisting ofCH—OH, C═O, C═S, S═O and SO₂; each of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is amoiety independently selected from the group consisting of hydrogen, acyclic or an acyclic, substituted or unsubstituted, branched orunbranched aliphatic group, a cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic group, asubstituted or unsubstituted, branched or unbranched acyl group, asubstituted or unsubstituted, branched or unbranched aryl group, asubstituted or unsubstituted, branched or unbranched heteroaryl group, xis an integer independently having the value between 1 and 10,inclusively, n is an integer independently having the value between 1and 3, inclusively, m is an integer independently having the valuebetween 0 and 20, inclusively, p is an integer independently having thevalue of 0 or 1, wherein if m=p=0, then R₂ is hydrogen, with the furtherproviso that if at least one of n or m has the value of 2, then R₃ andnitrogen in structure I form a moiety selected from the group consistingof:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, andeach * indicates the nitrogen atom in structure I.

In some embodiments, R₃ is a polyamine. In other embodiments, R₃ is aketal. In some embodiments, each of R₁ and R₂ in the general structure Iis independently any of substituted or unsubstituted, branched orunbranched alkyl or alkenyl groups having between 3 and about 20 carbonatoms, such as between 8 and about 18 carbon atoms, and between 0 and 4double bonds, such as between 0 and 2 double bonds.

In some embodiments, if each of n and m independently has the value of 1or 3, R₃ is any of the following moieties:

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structure I,where each H on any * position can be replaced to achieve the attachmentto the nitrogen atom in structure I.

According to some embodiments, compounds the general structure I mayhave each of R₄, R₅, R₆ and R₇ being hydrogen, each of Y and Z beingC═O, each of R₁ and R₂ being the same and each of X₁ and X₂ also beingthe same. Such compounds are represented by the general structure II(which is a sub-genus of the compound the general structure I):

wherein when n=p=0, R₂ is H.

In compounds of the general structure II, at least one X₁ is NH, or atleast one X₁ is O. Furthermore, in some embodiments, in compounds of thegeneral structure II, each R₁ is independently any of substituted orunsubstituted, branched or unbranched alkyl or alkenyl groups havingbetween 3 and about 20 carbon atoms, e.g., between 8 and about 18 carbonatoms, and between 0 and 4 double bonds, e.g., between 0 and 2 doublebonds. Furthermore, in some embodiments, if each of n and mindependently has the value of 1 or 3, R₃ is any of the followingmoieties:

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structureII, where each H on any * position can be replaced to achieve theattachment to the nitrogen atom in structure II.

In some embodiments, if at least one of n or m in the general structureII has the value of 2, then R₃ is either of the following moieties:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain,and each * shows a point of attachment of R₃ to the nitrogen atom instructure II.

According to embodiments of the invention, there are provided somespecific compounds that are species within either the general structureI or the general structure II, or both. Non-limiting examples of suchspecific compounds are any of the following lipids 1-87, or any isomerof each of compounds 1-87, or any combination of isomers for each ofcompounds 1-87:

Some of compounds 1-87 shown above were used to prepare some lipidcompositions. These compositions are described in detail in Example 9below, and are shown in Table 1 in Example 9. The numbers of thecompounds in the first column of Table 1 correspond to the numbers ofcompounds shown above.

According to other embodiments of the invention, the above-describedcompounds of the invention may be synthesized by reacting an aminocomponent with an unsaturated component, e.g., by the addition of theprimary amino group of the amino component to a double bond of theunsaturated component where the double is conjugated with anelectrophilic group such as, e.g., carbonyl. In general, the syntheticmethod includes reacting one equivalent of the amino component with oneor more equivalents of the unsaturated component. The amino componentcomprises a primary amine NH₂—R₃, a diamine, a polyamine or acombination thereof. The unsaturated component comprises of at least onefirst intermediate having the structure R₁—X₁—Y—(CR₄R₅)_(n)—Br and thesecond intermediate having the structure R₂—X₂—Z—(CR₆R₇)_(m)—Br, whereinin (CR₄R₅)_(n) and (CR₆R₇)_(m) portions of the structures, each R₄ isthe same or different, each R₅ is the same or different, each R₆ is thesame or different, and each R₇ is the same or different, wherein thefirst and the second intermediates are the same or different. In someembodiments, the first and/or the second intermediate(s) of theunsaturated component can be an acrylate or acrylamide.

In certain embodiments, all the amino groups of the amine NH₂—R₃, adiamine or a polyamine are fully reacted with the unsaturated componentto form tertiary amines. In other embodiments, not all the amino groupsare so reacted to form tertiary amines thereby resulting in primary orsecondary amines in the lipid molecule.

The synthesis of the compounds of the invention may be performed with orwithout solvent, and the synthesis may be performed at temperaturesranging between room temperature and about 100° C., for example, atabout 95° C. The reaction may be optionally catalyzed by adding an acid,a base or a metal. Those having ordinary skill in the art can select theoptimal conditions under which the synthesis is carried out, to choosean appropriate catalyst, if necessary, and to select the molar ratiobetween the amino component and the unsaturated component. For example,when the amino component is the primary amine NH₂—R₃, the molar ratiobetween the unsaturated component and the primary amine NH₂—R₃ can bebetween about 1:1 and about 6:1. The prepared lipids may be optionallypurified. The lipids may also be alkylated using an alkyl halide (e.g.,methyl iodide) or other alkylating agent.

In one exemplary non-limiting, embodiment the synthetic process can beillustrated by the following reaction scheme:

In the above-provided structures of the first and the secondintermediates comprising the unsaturated component, each of X₁ and X₂ isa moiety independently selected from the group consisting of O, S, N-Aand C-A, wherein A is selected from the group consisting of hydrogen anda C₁-C₂₀ hydrocarbon chain; each of Y and Z is a moiety independentlyselected from the group consisting of CH—OH, C═O, C═S, S═O and SO₂; eachof R₁, R₂, R₄, R₅, R₆ and R₇ is a moiety independently selected from thegroup consisting of hydrogen, a cyclic or an acyclic, substituted orunsubstituted, branched or unbranched aliphatic group, a cyclic oracyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic group, a substituted or unsubstituted, branched orunbranched acyl group, a substituted or unsubstituted, branched orunbranched aryl group, a substituted or unsubstituted, branched orunbranched heteroaryl group, and each of n and m is an integerindependently having the value between 1 and 3, inclusively.

In some embodiments, if at least one of n or m in the above-providedstructures of the first and the second intermediates of the unsaturatedcomponent has the value of 2, and the primary amine NH₂—R₃ is used asthe amino component, then R₃ in the above-provided structure of theprimary amine is either of the following moieties:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain,and each * shows a point of attachment of R₃ to the amino group in theprimary amine NH₂—R₃.

In some embodiments, each of R₁ and R₂ in the above-provided structuresof the first and the second intermediates of the unsaturated componentis independently any of substituted or unsubstituted, branched orunbranched alkyl or alkenyl groups having between 3 and about 20 carbonatoms, such as between 8 and about 18 carbon atoms, and between 0 and 4double bonds, such as between 0 and 2 double bonds.

In some embodiments, if in the above-provided structures of the firstand the second intermediates of the unsaturated component each of n andm independently has the value of 1 or 3, and the primary amine NH₂—R₃ isused as the amino component, R₃ in the above-provided structure of theprimary amine can be any of the following moieties:

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the amino group in the primaryamine NH₂—R₃, where each H on any * position can be replaced to achievethe attachment to the nitrogen atom in the primary amine NH₂—R₃.

The above described compounds of the invention may be used in thedelivery of biologically active or therapeutic agents to a subject, toan animal, or to a cell or a tissue in vitro or in vivo. In certainillustrative though non-limiting embodiments, the compounds may beparticularly suited to delivering negatively charged bioactive agents.For example, the amin-containing transfection compounds of the presentinvention may be used to delivery DNA, RNA, other polynucleotides, otheranions or polyanions to a subject or to a cell.

In some embodiments, the inventive lipids are combined with an agent toform transfection complexes, such as microparticles, liposomes ormicelles. The bioactive agent to be delivered (e.g., a polynucleotide, aprotein, a peptide or a small molecule) by these delivery vehicles maybe in the solid or liquid or dissolved form. The inventive lipids may becombined with other lipids, polymers, surfactants, cholesterol,carbohydrates, proteins, etc. to form the particles. These particles maybe combined with a pharmaceutically excipient to form pharmaceuticalcompositions.

The lipid synthesized as described above may be further purified by anyknown technique, such as by crystallization, chromatography,precipitation (e.g. repeated precipitations in diethyl ether, hexane oranother organic solvent) or distillation. The lipid may be also isolatedas a salt that can be formed when the lipid is reacted with an organicacid or inorganic acid. In some embodiments, if the lipid comprises thetertiary amine moiety, it can be alkylated with any alkylating agent,for example, alkyl halides such as methyl iodide to form a quaternaryammonium salt of the lipid. The anion associated with the quaternaryamine may be any pharmaceutically acceptable organic or inorganic anion.

In some embodiments, the synthetic process results in a mixture ofisomers having acrylic tails, with varying numbers and positions of theacrylate tails. Such mixtures can be used with or without furtherpurification, as desired. When an amine is not exhaustively alkylated,the resulting products may be further reacted with another electrophile,such as an acrylate or acrylamide optionally followed by furtherpurification.

In some embodiments, a library of different lipids can be prepared inparallel. To that end, a different amine and/or unsaturated componentcan be added to each vial in a set of vials or to each well of amulti-well plate. The array of reaction mixtures is incubated at atemperature and length of time sufficient to allow formation of thelipids to occur. The lipids may then be isolated and purified usingknown techniques followed by screening using high-throughput techniquesto identify lipids with a desired characteristic (e.g., solubility,ability to bind polynucleotides, ability to bind heparin, ability tobind small molecules, ability to form microparticles, ability toincrease transfection efficiency and the like).

Transfection Complexes

It is a further object of the presently disclosed embodiments to providenovel transfection complexes that are formulated to deliver abiologically active compound to a cell or a tissue in vitro, or to acell or tissue in an animal in vivo. The delivery of a biologicallyactive agent to cells or tissue as contemplated herein may be for theprovision of a therapeutic modality for the treatment of a disorder, ormay alternatively be provided during the course of conducting scientificresearch (e.g., as a research tool).

In some embodiments, a transfection complex as provided for herein mayinclude one or more amine-containing transfection agents formulated as alipid aggregate such that the biologically active agent can be deliveredto a cell or a tissue to affect a desired biological response. In someembodiments, a transfection complex may optionally include one or morehelper lipids. In an embodiment, a transfection complex may optionallyinclude one or more pegylated lipids. In an embodiment, a transfectioncomplex may optionally include one or more targeting moieties ortransfection enhancers.

In an embodiment, an a transfection complex may include anamine-containing transfection compound having the general structure I,or pharmaceutically acceptable salts thereof:

wherein each of X₁ and X₂ is a moiety independently selected from thegroup consisting of O, S, N-A and C-A, wherein A is selected from thegroup consisting of hydrogen and a C₁-C₂₀ hydrocarbon chain; each of Yand Z is a moiety independently selected from the group consisting ofCH—OH, C═O, C═S, S═O and SO₂; each of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is amoiety independently selected from the group consisting of hydrogen, acyclic or an acyclic, substituted or unsubstituted, branched orunbranched aliphatic group, a cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic group, asubstituted or unsubstituted, branched or unbranched acyl group, asubstituted or unsubstituted, branched or unbranched aryl group, asubstituted or unsubstituted, branched or unbranched heteroaryl group, xis an integer independently having the value between 1 and 10,inclusively, n is an integer independently having the value between 1and 3, inclusively, m is an integer independently having the valuebetween 0 and 20, inclusively, p is an integer independently having thevalue of 0 or 1, wherein if m=p=0, then R₂ is hydrogen, with the furtherproviso that if at least one of n or m has the value of 2, then R₃ andnitrogen in structure I form a moiety selected from the group consistingof:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, andeach * indicates the nitrogen atom in structure I.

In some embodiments, R₃ is a polyamine. In other embodiments, R₃ is aketal. In some embodiments, each of R₁ and R₂ in the general structure Iis independently any of substituted or unsubstituted, branched orunbranched alkyl or alkenyl groups having between 3 and about 20 carbonatoms, such as between 8 and about 18 carbon atoms, and between 0 and 4double bonds, such as between 0 and 2 double bonds.

In some embodiments, if each of n and m independently has the value of 1or 3, R₃ is any of the following moieties:

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structure I,where each H on any * position can be replaced to achieve the attachmentto the nitrogen atom in structure I.

According to some embodiments, transfection complexes containingamine-containing transfection compounds having the general structure Imay have each of R₄, R₅, R₆ and R₇ being hydrogen, each of Y and Z beingC═O, each of R₁ and R₂ being the same and each of X₁ and X₂ also beingthe same. Such compounds are represented by the general structure II(which is a sub-genus of the compound the general structure I):

wherein when n=p=0, R₂ is H.

In compounds of the general structure II, at least one X₁ is NH, or atleast one X₁ is O. Furthermore, in some embodiments, in compounds of thegeneral structure II, each R₁ is independently any of substituted orunsubstituted, branched or unbranched alkyl or alkenyl groups havingbetween 3 and about 20 carbon atoms, e.g., between 8 and about 18 carbonatoms, and between 0 and 4 double bonds, e.g., between 0 and 2 doublebonds. Furthermore, in some embodiments, if each of n and mindependently has the value of 1 or 3, R₃ is any of the followingmoieties:

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structureII, where each H on any * position can be replaced to achieve theattachment to the nitrogen atom in structure II.

In some embodiments, if at least one of n or m in the general structureII has the value of 2, then R₃ is either of the following moieties:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, each “HCC” symbolizes a hydrocarbon chain,and each * shows a point of attachment of R₃ to the nitrogen atom instructure II.

According to embodiments of the invention, transfection complexes mayinclude one or more amine-containing transfection compounds that arespecies within either the general structure I or the general structureII, or both. Non-limiting examples of such specific compounds are any ofthe following lipids 1-87 indicated above, or any isomer of each ofcompounds 1-87, or any combination of isomers for each of compounds1-87:

In some embodiments, the molar percentage of the amine-containingtransfection compound is from about 15% to about 50% of the lipidaggregate; in other embodiments, the molar percentage of the cationiclipid is from about 20% to about 40% of the transfection complex; insome embodiments, the molar percentage of the amine-containingtransfection compound is from about 25% to about 35% of the lipidaggregate; or, in some embodiments, the molar percentage of theamine-containing transfection compound is about 33% of the lipidaggregate. In some embodiments, the molar percentage of theamine-containing transfection compound is between about 15% and about35% of the lipid aggregate; in other embodiments, the molar percentageof the amine-containing transfection compound is between about 20% andabout 30% of the lipid aggregate; or in some embodiments, the molarpercentage of the amine-containing transfection compound isapproximately 25% of the lipid aggregate.

In some embodiments, a transfection complex may optionally include oneor more helper lipids. Illustrative though non-limiting examples ofhelper lipids suitable for use in the formulation of the presentlydescribed transfection complexes include cholesterols, cholesterolderivatives, sterols, including phytosterols, zoosterols and hopanoids,or any of the neutral or cationic lipids that are known to allow or tofacilitate the introduction of exogenous bioactive molecules to theinterior of a cell or of a tissue. In some embodiments, more than onehelper lipid may be used in the formulation of the transfectioncomplexes described herein. Exemplary though non-limiting neutral orcationic lipids contemplated for use in the preparation of the presentlydisclosed transfection complexes may include one or lipids selected fromthe following: BMOP(N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimunbromide), DDPES (Dipalmitoylphosphatidylethanolamine5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation ofcetyltrimethylammonium bromide (CATB) and DOPE), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE(dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine),DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC(Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine),DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC(dioleoydimethylammonium chloride), DOSPER(1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA(N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride), DDAB(didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate),DOTAP.Cl, DC-chol(3,β-N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA(2-(sperminecarboxamido)ethyl)-N,N-dimethy-lammonium trifluoroacetate),DC-6-14 (O,O′-Ditetradecanoyl-N-(alphatrimethylammonioacetyl)diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE(dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP(1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA(2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminiumtrifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE(N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME(N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammoniumiodide), GAP-DLRIE:DOPE(N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide/dioleyl phosphatidylethanolamine), DPPC(Dipalmitoylphosphatidylcholine), DOPG(1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl),N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivativesthereof); phosphotidylethanolamine (and derivatives thereof);phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPEdipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, O-Choi (3beta[1-ornithinamidecarbamoyl]cholesterol), POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; piperazine-based cationic lipids,phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG(dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;phosphatidylserine (and derivatives thereof); phosphatidylserines, suchas dioleoyl- or dipalmitoylphosphatidylserine; diquaternary ammoniumsalts such as N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,2-ethanediamine(TmedEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,3-propanediamine(PropEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,6-hexanediamine(HexEce), and their corresponding N,N′-dicetyl saturated analogues(TmedAce, PropAce and HexAce), diphosphatidylglycerols; fatty acidesters; monocationic transfection lipids such as1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol;1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol;1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol;1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βOH-sterolsas well as derivatives thereof phosphatidyl choline or commerciallyavailable cationic lipid mixtures such as, for example, LIPOFECTIN®CELLFECTIN® (1:1.5 (M/M) formulation of N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS) anddioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXN™,TRANSFAST™, TRANSFECTAM®, TRANSMESSENGER®, vectamidine(3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine (a.k.a.diC14-amidine), OLIGOFECTAMINE®, among others. Also contemplated are anymixtures of combination of the above listed helper lipids. The followingpatent documents, patent applications, or references are incorporated byreference herein in their entirety and in particular for theirdisclosure of transfection agents containing cationic and neutral helperlipids which may be used to comprise the transfection complexes of thepresent invention.

The molar percentage of the helper or neutral lipid is between about 60%and about 85% of the lipid aggregate; in some embodiments, the molarpercentage of the helper or neutral lipid is between about 70% and about80% of the lipid aggregate; or, in some embodiments, the molarpercentage of the helper or neutral lipid is between about 70% and about75% of the lipid aggregate.

In some embodiments, a transfection complex may include one or morePegylated lipids. Pegylated lipids suitable for use in the preparationand formation of transfection complexes disclosed herein can be anylipid or mixture of lipids that are compatible with the formation oftransfection complexes described herein, and with the administrationthereof to an animal or to a human in vivo, or to tissues or cells invitro. The pegylated lipids used in the present invention include a PEGpolymer having a molecular weight between about 250 daltons and about12,000, or in some embodiments, about 350 daltons and about 6,000daltons, or, in some embodiments, between about 500 daltons and about1,000 daltons, or, in some embodiments, between about 1,000 daltons andabout 2,000 daltons, or, in some embodiments, between about 2,000daltons and 5,000 daltons. More specifically, suitable Pegylated lipidsinclude phosphatidylethanolamine (PE) based pegylated lipids such as,for example,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-MW] where MW refers to average MW of the polyethylene glycolmoiety. Such dimyristoyl-PEG-PE lipids are commonly designated 14:0 PEG(MW) PE. The average MW of the polyethylene glycol moiety can be 25,350, 550, 750, 1000, 2000, 3000, 5000, 6000, 8000 or 12000, for example.The fatty acid chains of the phosphatidylethanolamine based pegylatedlipids may include, for example, a 1,2-dioleoyl group such as for 18:1PEG (MW) PE, a 1,2-dipalmitoyl group such as for 16:0 PEG (MW) PE, or a1,2-distearoyl-group such as for 18:0 PEG (MW) PE. Furtherphosphatidylethanolamine (PE) based pegylated lipids include, forexample,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[MOD(polyethyleneglycol)-MW], also referred to as DSPE-MOD PEG(MW) wherein MOD refers toa functional moiety such as an amine, biotin, carboxylic acid, folate,maleimide, PDP, or carboxyfluorescein moiety. The MW may be 2000 or5000, for example. Pegylated lipids for the embodiments described hereinalso include ceramide based pegylated lipids such as, for example,N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)MW]},designated C8 PEG (MW) ceramide, where MW is 750, 2000, or 5000, forexample. Alternatively, the fatty acid moiety may have an N-palmitoyl(C16) group such as for C16 PEG (MW) ceramide.

In some embodiments, the molar percentage of the pegylated lipid isbetween about 0.5% and 15% of the transfection complex; in someembodiments, the molar percentage of the pegylated lipid is betweenabout 1% and about 10% of the transfection complex; or in someembodiments, the molar percentage of the pegylated lipid isapproximately 1% or 5% of the transfection complex.

In some embodiments, the molar percentage of amine-containingtransfection compound: helper lipid:pegylated lipid of the transfectioncomplex ranges from 15:84:1 to 15:75:10, from 20:79:1 to 20:70:10, from25:74:1 to 25:65:10, from 30:69:1 to 30:60:10, from 40:59:1 to 40:50:10,or from 50:49:1 to 50:40:10. In some embodiments, the molar percentageof amine-containing transfection compound: helper lipid:pegylated lipidof the transfection complex ranges from 10-90:7-35:5-70, from15-85:5-35:8-50, from 30-85:5-35:8-50, from 35-70;10-30:15-45, from40-65:15-25:20-40, from 50-60:18-22:25-35, from 50-55:19-21:27-30, orfrom 51-53:20-20.5:28-29. Of course, it will be readily appreciated bythe skilled practitioner that alternative ratios may be employed, andoptimizing the ratios of such formulations is well within the skilllevel of such a person, without requiring undue experimentation.

Further non-limiting embodiments of the present invention providemethods for delivering a bioactive agent, such as, e.g., a polyanion, apolynucleotide or a polypeptide into a cell or cells, or into a tissue,wherein the method includes forming a lipid aggregate, such as aliposome, comprising one or more of the amine-containing transfectioncompounds described above, optionally with one or more helper lipidsand/or one or more pegylated lipids, and contacting the lipid aggregatewith the bioactive agent to form a neutral or positively chargedbioactive agent-lipid aggregate complex, and incubating the complex witha cell or a tissue in vitro, or administering the resulting transfectioncomplex to an animal or to a human, optionally as a therapeuticcomposition. Useful bioactive agents contemplated for suchadministration include proteins, peptides and nucleic acids, such as DNAor RNA.

In some embodiments, the transfection complexes may include one or morebiologically active agents to be delivered to a cell or to a targettissue in vitro or in vivo. Suitable biologically active agents mayinclude any molecule that is capable of forming a transfection complexwith the presently described amine-containing transfection reagents andthat elicits a biological response when delivered to the interior of acell or cells or to a tissue in vivo or in vitro. Biologically activeagents contemplated for use in the presently described embodiments maybe cationic, neutral or anionic agents. By way of non-limiting example,exemplary biologically active agents suitable for formulation in atransfection complex may include, though are not limited to; nucleicacids (including but not limited to single or double stranded linear orcircular DNA molecules including cDNA molecules, single or doublestranded RNA molecules, small interfering RNA (siRNA) molecules, smallhairpin RNA (shRNA) molecules, microRNA (miRNA) molecules,oligonucleotides, anti-sense oligonucleotides, sense oligonucleotides),polypeptides, antibodies, oligopeptides, therapeutic peptides or proteinmolecules, peptide nucleic acids (PNAs), cationic, anionic or neutralorganic molecules or drugs, in addition to pharmaceutically acceptablesalts thereof.

In certain non-limiting illustrative embodiments of the invention,transfection complexes and methods are provided that use the compoundsof the present invention to deliver nucleic acid molecules into cells ortissues in vitro or in vivo, including the delivery of RNA interferencemolecules (RNAi) or small interfering RNA molecules (siRNA, shRNA ormiRNA) into cells for inhibition of gene expression.

In certain non-limiting illustrative embodiments, transfection complexesand methods are provided that use the compounds of the present inventionto deliver mRNA molecules into a cell or a tissue in vivo or in vitro topromote the expression of a specific protein or proteins are alsoprovided.

In other non-limiting illustrative embodiments of the invention,transfection complexes and methods are provided that use the compoundsof the present invention to deliver DNA molecules (including cDNAmolecules) into a cell or a tissue in vivo or in vitro to promote theexpression of a specific protein or proteins or to synthesize specificRNA molecules, including but not limited to mRNA molecules or RNAi ormiRNA or shRNA molecules are also provided.

In some embodiments, the transfection complexes described herein mayoptionally include one or more fusogenic or cell-penetrating peptides. Afusogenic or cell-penetrating peptide is any peptide molecule that iscapable of promoting the fusion of a lipid-containing complex to a cellmembrane (either a plasma membrane or an endosomal membrane). A varietyof fusogenic or cell-penetrating peptides are known in the art and it iswell within the skill level of a practitioner to identify suitablefusogenic or cell-penetrating peptides and condition for the use thereofin the present invention without undue experimentation.

In some embodiments, the transfection complexes described herein mayoptionally include one or more transfection helpers or targetingmoieties. A targeting moiety may be a peptide, a modified peptide, anantibody, a modified antibody, a receptor molecule, a modified receptormolecule, a single or a double stranded nucleic acid molecule, amodified single or double stranded nucleic acid molecule, a peptide ornucleic acid aptamer, a modified peptide or nucleic acid aptamer, anorganic molecule, a polysaccharide, or any other molecule that iscapable of targeting a transfection complex to specific tissue or celltype for targeted delivery of a biologically agent thereto, such as willbe readily apparent to have having ordinary skill level in the art. Insome embodiments, modification of a peptide, an antibody, a nucleicacid, an aptamer, and the like may include conjugating the peptide,antibody, nucleic acid, aptamer, and the like to a PEG moiety.Alternatively, modification of a peptide, an antibody, a nucleic acid,an aptamer, and the like may include conjugating the peptide, antibody,nucleic acid, aptamer, and the like to a PEG-lipid moiety A variety oftargeting moieties are widely known to those skilled in the art, and allare contemplated for use with the presently described embodiments,without limitation.

In some embodiments, the transfection complexes provided for herein maybe stable for up to 1 year and may either be contacted with the cells ortissues to be transfected, or be administered to an animal or to a humanimmediately or shortly after being formed, or optionally may stored fora period of time prior to being contacted with the cells or tissues, orbeing administered to an animal or a human. The transfection complexesare stable and may be stored for a time period of at least 30 minutes,at least 45 minutes, at least 1 hour, at least 2 hours, at least 3hours, at least 4 hours, at least 5 hours, at least 10 hours, at least15 hours, at least 20 hours, at least 24 hours, at least 48 hours, atleast 72 hours, at least 5 days, at least 7 days, at least 14 days, atleast 28 days, at least 1 month, at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months or at least 1year at room temperature, or at a temperature greater than freezing, upto about room temperature. It is to be understood that the formulationdescribed herein may include one or more stabilizing agents,preservatives, buffers, etc, that aid in the long-term stabilization andstorage of bioactive formulation, such as will be readily understood bythe skilled practitioner of the biological and pharmaceutical arts, andwithout requiring undue experimentation to achieve. It is alsounderstood, that the storage period can be between any of these timeperiods, for example between 31 minutes and 1 hour or between 1 hour and24 hours.

Optionally, the bioactive agent-lipid aggregate complex is stored for aperiod prior to being contacted with the cell or cells. Thepolyanion-lipid aggregate complex is stable and can be stored for a timeperiod of at least 45 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, atleast 15 hours, at least 20 hours, at least 24 hours, at least 48 hours,at least 72 hours, at least 5 days, at least 7 days, at least 14 days,at least 28 days, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months or atleast 1 year, or for a time period between any of these time periods.

The present invention is particularly suited to deliver RNAi components,including siRNA, short hairpin RNA (shRNA), microRNAs (miRNA) and smalltemporally regulated RNA (stRNA), which optionally are chemicallymodified, to cells or to tissues in vitro or in vivo.

The delivery methods employing the transfection complexes of the presentinvention or mixtures thereof can be applied to cells in vitro, ex vivo,and in vivo, particularly for transfection of eukaryotic cells ortissues including animal cells, human cells, non-human animal cells,insect cells, plant cells (including algae), avian cells, fish cells,mammalian cells and the like.

In some embodiments, the bioactive agent that is to be delivered intothe cell is contacted with lipid aggregates of this invention to form atransfection complex comprising a bioactive agent-lipid aggregatecomplex. The target cell or cells or the target tissues are thenincubated with the complex, or, for in vivo applications, the complex isadministered to the organism by an appropriate route (e.g., intravenous,intramuscular, subcutaneous, transdermal, transmucosal, etc) so that thecomplex contacts the target cells or tissue. Methods for the applicationof transfection complexes to cells or tissue in vitro or to tissues oran animal in vivo are widely known in the art, and the use of suchmethods is contemplated herein without limitation for the administrationof the presently described lipid transfection complexes to cell, tissuesor an animal. It is well within the ability of the practitioner havingordinary skill level in the art to adapt these methods for use with thepresently described compositions without departing from the spirit andthe scope of the invention described herein. The compounds of may alsobe conjugated to or mixed with or used in conjunction with a variety ofuseful molecules and substances, also referred to as transfectionhelpers or targeting moieties, such as proteins, peptides, growthfactors, antibodies, nucleic acids, aptamers, or modified versionsthereof (such as, e.g., conjugating said transfection helpers ortargeting moieties to PEG or PEG-lipids) and the like to enhancecell-targeting, uptake, internalization, nuclear targeting andexpression, all of which are likewise within the skill level of theskilled practitioner.

A further embodiment provides a method of transfecting a cell or tissuewith a nucleic acid in vivo wherein the method comprises forming a lipidaggregate, such as a liposome, comprising one or more amine-containingtransfection compounds, one or more pegylated lipids and optionally oneor more helper lipids, contacting the lipid aggregate with the nucleicacid to form a neutral or positively charged lipid aggregate-nucleicacid complex, and administering the lipid aggregate-nucleic acid complexto the cells or tissues in vitro or to an organism so that the complexcontacts the target cells or tissue. Administration of the lipidaggregate-nucleic acid complex can be achieved orally, intravenously, orby subcutaneous or intramuscular injection or applied topically to thetissue as further described below.

Optionally, the bioactive agent-lipid aggregate complex is stored for aperiod prior to being contacted with the cell or cells for transfection.The polyanion-lipid aggregate complex is stable and can be stored for atime period of at least 30 minutes, at least 45 minutes, at least 1hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5hours, at least 10 hours, at least 15 hours, at least 20 hours, at least24 hours, at least 48 hours, at least 72 hours, at least 5 days, atleast 7 days, at least 14 days, at least 28 days, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months or at least 1 year, or for a time period between anyof these time periods.

In another embodiment, transfection complexes of the present invention(approximately between 5 μl and 2000 μl) are provided in the wells of amultiwell plate. Bioactive molecules to be delivered into target cellsare selected and added to the wells to form polyanion-lipid aggregatecomplexes, which are subsequently contacted with the target cells invitro or in vivo. The lipid aggregates can have the same composition andconcentration in each well, or the lipid aggregate composition and/orconcentration can vary from well to well (for example, the amount ofpegylation in the lipid aggregate can vary across the wells to determinethe range for delivery and transfection). Where the bioactive agents arenucleic acids such as RNAi, the nucleic acids can be added to the wellsand optionally stored before contacting with the target cells.

The methods of this invention optionally comprise the step of contactingthe one or more amine-containing transfection compounds with one or morehelper lipids and one or more pegylated lipids before or at the sametime as contacting the bioactive agent with the one or moreamine-containing transfection compounds to form lipid aggregatesencapsulating the bioactive agent. The methods also optionally compriseforming the lipid aggregates into liposomes prior to contact with thebioactive agent. In further embodiments, the liposomes are formed bymicrofluidization, extrusion or other means known in the art. In someembodiments, the bioactive agents may include nucleic acid moleculessuch as DNA or RNA that inhibit or promote the expression of a targetgene. In some embodiments, the bioactive agent associates with atranscript of the gene to effect inhibition thereof. In someembodiments, the bioactive agent is a nucleic acid, such as a DNA anmRNA, RNAi, siRNA, shRNA, miRNA or stRNA, and is optionally chemicallymodified.

In another embodiment, the invention also provides medicaments preparedby combining liposome comprising one or more amine-containingtransfection compounds optionally with one or more helper lipids,optionally with one or more pegylated lipids and optionally with one ormore targeting moieties or transfection helpers (as described above) ora salt thereof, with a bioactive agent such as, e.g., a nucleic acid,wherein introduction of the bioactive agent into a cell or tissuemodulates expression of one or more target genes therein therebyeffecting at least one biological response and/or at least onetherapeutic benefit. Optionally the medicament further comprises anadditional excipient or pharmaceutical carrier, such as will be readilyapparent to the practitioner having ordinary skill level in thepharmaceutical and/or the medical arts. In some embodiments, thebioactive agent may be a nucleic acid, such as, e.g., a DNA or RNA. Insome embodiments, the nucleic acid is mRNA, RNAi, siRNA, shRNA, miRNA orstRNA, and is optionally chemically modified. In some embodiments,medicaments or pharmaceutical preparations are provided for treatment ofa disease, condition, or disorder that relates to the expression of oneor more genes in a cell or a tissue. Formulation of pharmacologicallyacceptable medicaments is known in the art. Administration of themedicament delivers an effective amount of the polyanion, for exampleRNA or a DNA, to the cells or tissue associated with the disease,condition, or disorder to provide amelioration of the disease, conditionor disorder. By way of non-limiting examples, such medicaments can beadministered orally, intravenously, or by subcutaneous or intramuscularinjection or applied topically to the tissue as further described below.

The invention also provides kits for the preparation of one or moretransfection complexes of the presently described invention. Such kitsmay, for example, comprise one or more liposomal compositions of thisinvention. Such kits typically comprise a carrier, such as a box,carton, tube or the like, having in close confinement therein one ormore containers, such as vials, tubes, ampules, bottles, and the like,wherein containers contain one or more amine-containing transfectioncompounds, optionally with one or more helper lipids, optionally withone or more pegylated or cationic lipids (or acceptable salts thereof)in accordance with the embodiments described above, or a liposomalcompositions of the present invention. The kits encompassed by thisaspect of the present invention may further comprise one or moreadditional components (e.g., reagents and compounds) necessary orbeneficial for carrying out one or more particular applications of thecompositions of the present invention. In some embodiments, the kit mayoptionally contain one or more multiwell plates suitable for holding thelipid aggregates or transfection complexes of the present invention. Infurther examples, the kits may also contain one or more componentsuseful in carrying out a desired transfection of cells. In yet furtherexamples, the kit may also contain one or more components useful incarrying out diagnosis, treatment or prevention of a particular diseaseor physical disorder (e.g., one or more additional therapeutic compoundsor compositions, one or more diagnostic reagents). In general kits mayalso contain one or more buffers, positive or negative control samples,carriers or excipients, and the like, one or more additionalcompositions of the invention, one or more sets of instructions, and thelike.

Methods of encapsulating a bioactive agent, such as a nucleic acid, aDNA, and RNA, or the like, in a transfection complex (i.e., a lipidaggregate-bioactive agent complex) are provided as some embodiments.Such methods may include forming a lipid aggregate comprising one ormore amine-containing transfection compounds, such as those representedin structures I and II and in formulae 1-87, one or more helper lipids,one or more pegylated lipids, and optionally one or more transfectionhelpers or targeting moieties under any one or more of the followingconditions:

a1) mixing one or more amine-containing transfection compounds, at leastone helper lipid, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, in an alcohol/aqueous solutionwherein the alcohol concentration is <50%;

a2) mixing one or more amine-containing transfection compounds, at leastone helper, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, in a molar percentage such that theone or more amine-containing transfection compounds are present at15%-50%;

a3) mixing one or more amine-containing transfection compounds, at leastone helper lipid, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, in a molar percentage such that thePegylated lipids are present at <50%; and

a4) mixing one or more amine-containing transfection compounds, at leastone helper lipid, optionally more than one helper lipid and one or morepegylated lipids, or a salt thereof, wherein the pegylated lipid has apolyethylene glycol molecular weight of about 2000-12000 and a fattyacid chain length of C₆-C₂₀ alkyl, or C₁₀-C₂₀ alkenyl; and complexingthe lipid aggregate in an alcohol/aqueous solution with the bioactiveagent to form a transfection complex, wherein the alcohol concentrationis <50%, preferably less than 40%. In some embodiments, the methodincludes a1) and a2), a2) and a3), a1) and a3), a2) and a4), a3) anda4), a1) and a4), or a1)-a4), for example. In some embodiments, thealcohol is a C1-C4 alcohol. In some embodiments, the alcohol is ethanol.In some embodiments, the alcohol is a pharmaceutically acceptablealcohol such as an alcohol that is liquid at about room temperature, forexample, ethanol, propylene glycol, 2-(2-ethoxyethoxyl)ethanol(Transcutol™), benzyl alcohol, glycerol, polyethylene glycol 200,polyethylene glycol 300, polyethylene glycol 400 or a mixture thereof.In some embodiments, the alcohol for mixing is different than thealcohol for complexing.

Transfection complexes of embodiments described herein may beadministered via the following routes for in vivo administration, forexample, intravenous, intradermal, intraarterial, intraperitoneal,intralesional, intracranial, intraarticular, intraventricular,intraprostatica, intrapleural, intratracheal, intranasal, intravitreal,intravaginal, intrauterine, intrarectal, topical, intratumoral,intrathecal, intramuscular, subcutaneous, subconjunctival,intravesicular, mucosal, intrapericardial, intraumbilical, intraocular,oral, topical, local, via inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, or byother methods or any combination of the foregoing as would be known toone of ordinary skill in the art (Remington's Pharmaceutical Sciences,18th Ed., Mack Publishing Co. 1990).

A dosage for ex vivo or in vivo use is from 0.01 μg to 1 g/kg of bodyweight, 0.1 μg to 0.1 g/kg of body weight, 1 μg to 0.01 g/kg of bodyweight, 10 μg to 0.01 g/kg of body weight, 0.1 mg to 10 mg/kg of bodyweight, or ranges between and including any of 0.1 mg, 0.25 mg, 0.5 mg,1.0 mg, 1.5 mg, 2 mg, and 10 mg/kg of body weight. Administration may beonce or more per day, week, month or year. Administration may be inbolus form. Generally, an effective amount of the lipoplexes ofembodiments herein is an amount sufficient to reduce expression of thetargeted gene and results in an extracellular concentration at thesurface of the target cell of from 100 pM to 1 μM, or from 1 nM to 100nM, or from 2 nM to about 25 nM, or to about 10 nM. The amount requiredto achieve this local concentration will vary depending on a number offactors including the delivery method, the site of delivery, the numberof cell layers between the delivery site and the target cell or tissue,whether delivery is local or systemic, etc. The concentration at thedelivery site may be considerably higher than it is at the surface ofthe target cell or tissue.

In some embodiments for in vivo administration, transfection complexesare prepared at a final concentration of 0.5-1 mg/ml. The ratio of lipidaggregate to nucleic acid is between about 0.7:1 and 1.3:1 (v:w)depending upon the targeted organ. One of ordinary skill in the art, inlight of the teachings herein, is able to test various ratios of lipidaggregate to nucleic acid for in vivo administration. For example, for a1 mg/ml lipoplex in 5% glucose, administration may be intravenousthereby targeting the lung, kidney, liver, tumor, or spleen (using50-200 μl), intraperitoneal thereby targeting a tumor or inflammation(using 100 l), intranasal thereby targeting the lung (using 50 μl),intratumoral or retro-orbital (local) thereby targeting the eye, atumor, knee-joint, or the ear (using 10-100 μl), intracerebral (local)thereby targeting the brain (using 0.5-5 μl), intrathecal therebytargeting the spinal cord (using 10 μl), or hydrodynamic therebytargeting the liver, kidney, or virus (using 0.8-2.5 ml), for example.

Methods for Screening Tissue-Specific Delivery

Further embodiments described herein provide for methods to screen largenumbers of transfection compounds for tissue-biased delivery in vivo.Such methods may include preparing a plurality of transfection complexescontaining a compound that readily facilitates the detection of a markerin combination with a test transfection compound, delivering each of theplurality of transfection complexes to a test animal, and detecting themarker.

In some embodiments, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of transfectioncomplexes, each transfection complex having at least one testtransfection compound in combination with at least one nucleic acid thatfacilitates detection of delivery to a tissue. The nucleic acid may bean RNA molecule or a DNA molecule that encodes a protein that can bedirectly detected (such as, e.g., Green Fluorescent Protein (GFP), redFluorescent Protein, luciferase, or the like), or encode a protein thateffects expression of a protein that can be directly detected.

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes Green Fluorescent Protein. Each unique transfection complex maybe delivered either intravenously, subcutaneously, or to a tissue to atest animal, such as a mouse. After a predetermined amount of time,tissues from the mouse may be harvested and the expression of GFP invarious tissues may be detected by gross examination, histologicalexamination or by molecular detection (PCR, Western blotting, or thelike) to determine which to tissue or tissues transfection complexescontaining specific transfection compounds are delivered to.

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes Luciferase. Each unique transfection complex may be deliveredeither intravenously, subcutaneously, or to a tissue to a test animal,such as a mouse. After a predetermined amount of time, tissues from themouse may be harvested and the expression of Luciferase in varioustissues may be detected by gross examination, histological examinationor by molecular detection (PCR, Western blotting, or the like), orimaged in-vivo using the IVIS® Imaging System (Caliper).

In an embodiment, a method for screening tissue-biased delivery of atransfection complex may include preparing a plurality of uniquetransfection complexes, each transfection complex having at least onetest transfection compound in combination with an mRNA or a cDNA thatencodes a specific transcription factor. Each unique transfectioncomplex may be delivered either intravenously, subcutaneously, or to atissue to a transgenic animal that expresses a reporter gene (such as,e.g., luciferase) under the control of the specific transcriptionfactor. After a predetermined amount of time, tissues from thetransgenic animal may be harvested and the expression of reporter genein various tissues may be detected by gross examination, histologicalexamination or by molecular detection (PCR, Western blotting, or thelike). If the reporter gene is luciferase, detection may be accomplishedin-vivo using the IVIS® Imaging System (Caliper).

In one non-limiting embodiment, a method for screening tissue-biaseddelivery of a transfection complex may include preparing a plurality ofunique transfection complexes, each unique transfection complexcontaining at least one unique test transfection compound in combinationwith an mRNA or DNA molecule that encodes Cre recombinase. The pluralityof unique transfection complexes may also optionally include one or moretransfection enhancers, one or more helper lipids, one or more pegylatedlipids or one or more targeting moieties as described above andincorporated herein. Each of the unique transfection complexes may bedelivered either intravenously (for example by tail vein injection),subcutaneously or via intra-tissue injection to a transgenic mouse thatexpresses a reporter gene in the presence of Cre recombinase. In anexemplary embodiment, each of the unique transfection complexes may bedelivered to a transgenic mouse bearing a loxP-STOP-loxP-Luciferasegene, such as, e.g. any of the 12956(B6)-Gt(ROSA)26 transgenic mousestrains, in which the firefly luciferase gene is inserted into theGt(ROSA)26Sor locus. In this transgenic line, the luciferase gene isblocked by a loxP-flanked STOP sequence located in between theluciferase transgene and its promoter. In the presence of Crerecombinase (i.e., in tissues to which the Cre-mRNA or DNA containingtransfection complexes are delivered) the STOP sequence is excised andluciferase is expressed.

To determine which tissues Cre luciferase is expressed in (and hence towhich tissues each of the unique test transfection compound ispreferentially delivered), expression of the luciferase transgene may beaccomplished according to any of the widely-used techniques forassessing gene expression known to the skilled artisan (such as PCR,Northern blotting, Western blotting, or the like or directly measuringluciferase activity). In some preferred embodiments, the transgewholemount excised tissues or histologic sections may be examined, forexample, using the IVIS® In Vivo

III. EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Example 1 Synthesis of 2-Bromo-N-Dodecyl Acetamide

The title intermediate was synthesized according to the followinggeneral reaction scheme:

In a round bottom flask with pressure-equalizing addition funnel, to 200mL of dichloromethane was added 1.5 g (7.431 mmol, 1.05 eq.) ofbromoacetyl bromide and the mixture was stirred under nitrogen. Theflask was cooled to about 0° C. using an ice bath. 1.31 g (7.077 mmol)of dodecylamine acid 0.716 (983 μL, 7.077 mmol) of triethylamine weredissolved in 100 mL of dichloromethane. This solution was transferred tothe addition funnel, and bromoacetyl bromide solution was slowly addedwithin approximately 1 hour. The reaction mixture was stirred for aboutanother 1 hour at about 0° C. slowly warming to room temperature byletting the ice melt.

The resulting reaction mixture was transfer to a separatory funnel andextracted with 100 mL of saturated sodium bicarbonate solution. Theaqueous layer was washed with 50 mL of dichloromethane, and the combinedorganic layers were washed with 100 mL of saturated sodium bicarbonatesolution. Finally, the water layer was washed with 100 mL ofdichloromethane. The combined organic layers were dried over sodiumsulfate, filtered to remove the sodium sulfate, and the mixture wasconcentrated by rotary evaporator to give about 2.022 g (93.3% yield) ofpure product. Molecular weight calculated: 305.14, molecular weightobserved: 306.25.

Example 2 Synthesis Of Tetradecyl-2-Bromoacetate

The title intermediate was synthesized according to the followinggeneral reaction scheme:

3 g (1.3 mL, 14.862 mmol, 1.05 eq.) of bromoacetyl bromide weredissolved in 50 mL of dichloromethane. The flask was cooled to about 0°C. using an ice bath. 3.03 g (14.154 mmol) of tetradecanol weredissolved in 50 mL of dichloromethane and added to the above mixtureslowly via a pipettee, followed by adding about 5 drops of concentratedsulfuric acid and stirring at about 0° C. for about 30 minutes, then atroom temperature overnight.

About 150 mL of saturated sodium bicarbonate solution were added andstirred rapidly for about 30 minutes. The mixture was then transferredto a separatory funnel and the organic layer is collected. The aqueouslayer was washed with about 100 mL of dichloromethane. The combinedorganic layers were dried over sodium sulfate, filtered and concentratedto give 4.095 g (89.7% yield) of product.

No mass peak was observed. To confirm the structure a test reaction wasset up as follows: 4.6 mg (0.043 mmol) of benzylamine, 17 mg (23 μL,0.130 mmol, 3 eq.) of N,N-Diisopropylethylamine and 22 mg (0.065 mmol,1.5 eq.) of tetradecyl-2-bromoacetate were dissolved in 1 mL of DMF andplaced in the microwave at about 160° C./200 W for 10 minutes. Theresulting reaction mixture was analyzed by LCMS. Molecular weightcalculated: 361.56, molecular weight observed: 362.42.

Example 3 Synthesis Of Pentadecyl-4-Bromobutanoate

The title intermediate was synthesized according to the followinggeneral reaction scheme:

4-bromobutyl chloride (1 eq.) was dissolved in dichloromethane (50 mL)and the reaction flask was cooled to about 0° C. using an ice bath. Tothe above mixture, 1-pentadecanol (1 eq.) in dichloromethane (50 mL) wasadded slowly using a pressure-equalizing addition funnel over a periodof about 30 min To the resulting above mixture, a catalytic amount (5drops) of concentrated sulfuric acid was added. The reaction mixture wasstirred at about 0° C. for about 30 minutes and then stirred at roomtemperature for four hours. The reaction mixture was transferred to aseparatory funnel and the organic layer was washed with saturated sodiumbicarbonate (×3), water (×2), brine (×1). The organic layer was driedover sodium sulfate, filtered and concentrated to give the desiredproduct, pentadecyl-4-bromobutanoate as colorless oil (88% yield).

No mass peak was observed. The structure of the expected product wasconfirmed via a test reaction: benzylamine (1 eq.), N-methylmorpholine(1 eq.), pentadecyl-4-bromobutanoate (0.9 eq.) were added to a driedheavy walled pyrex tube containing DMF (1 mL). The reaction mixture wasexposed to microwave irradiation (200 W) for about 10 min at about 170°C. After the irradiation, the reaction mixture was allowed to coolthrough an inbuilt system in the instrument until the temperature hadfallen below about 30° C. The resulting reaction mixture was analyzed byLCMS. Molecular weight calculated: 403.64, molecular weight observed:404.50.

Example 4 Synthesis of Tetradecyl2-[3-Methylaminopropyl-(2-oxo-2-tetradecoxy-ethyl)amino]Acetate

The title compound was synthesized according to the following generalreaction scheme:

20 mg (0.227 mmol) of N-methylpropane-1,3-diamine, 160 mg (0.477 mmol,2.1 eq.) of tetradecyl-2-bromoacetate synthesized as described inExample 2 above, and 176 mg (237 μl, 1.361 mmol, 6 eq.) ofN,N-diisopropylethylamine were dissolved in 1 ml of dimethylformamideand heated to about 60° C. overnight. The resulting mixture was purifiedby preparative HPLC. The fractions containing the title compound werecollected and characterized by LCMS. Molecular weight calculated:596.55, molecular weight observed: 597.76.

Example 5 Synthesis of (a)2-[2-[2-[2-[bis[2-(Dodecylamino)-2-oxo-ethyl]amino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]ethylamino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]-N-dodecyl-acetamideand (b)2-[2-[2-[2-[bis[2-(Dodecylamino)-2-oxo-ethyl]amino]ethylamino]ethylamino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]-N-dodecyl-acetamide

The mixture of the two title compounds was synthesized according to thefollowing general reaction scheme, showing a 4-tail title compound (a)and a 5-tail title compound (b):

14.6 mg (0.1 mmol) of N,N′-bis(2-aminoethyl)ethane-1,2-diamine, 159 mg(0.52 mmol, 5.2 eq.) of 2-bromo-N-dodecyl acetamide obtained asdescribed in Example 1 above, and 129 mg (174 μl, 1 mmol, 10 eq.) ofN,N-diisopropylethylamine are dissolved in 1 ml DMF and heated to about60° C. overnight. The resulting mixture is purified by preparative HPLC.

2-[2-[2-[2-[bis[2-(dodecylamino)-2-oxo-ethyl]amino]ethylamino]ethylamino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]-N-dodecyl-acetamide(4-tail compound shown above) and2-[2-[2-[2-[bis[2-(dodecylamino)-2-oxo-ethyl]amino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]ethylamino]ethyl-[2-(dodecylamino)-2-oxo-ethyl]amino]-N-dodecyl-acetamide(5-tail compound shown above) were collected and characterized by LCMS.

For the 4-tail compound: molecular weight calculated is 1046.99,molecular weight observed was 1048.28. For the 5-tail compound:molecular weight calculated is 1272.20, molecular weight observed was1273.50.

Example 6 Synthesis of Pentadecyl-4-Bromobutanoate

The title intermediate was synthesized according to the followinggeneral reaction scheme:

4-bromobutyl chloride (1 eq.) was dissolved in dichloromethane (50 mL)and the reaction flask was cooled to about 0° C. using an ice bath. Tothe above mixture, 1-pentadecanol (1 eq.) in dichloromethane (50 mL) wasadded slowly using a pressure-equalizing addition funnel over a periodof about 30 min. To the resulting above mixture, a catalytic amount (5drops) of concentrated sulfuric acid was added. The reaction mixture wasstirred at about 0° C. for about 30 minutes and then stirred at roomtemperature for four hours. The reaction mixture was transferred to aseparatory funnel and the organic layer was washed with saturated sodiumbicarbonate (×3), water (×2), brine (×1). The organic layer was driedover sodium sulfate, filtered and concentrated to give the desiredproduct, pentadecyl-4-bromobutanoate as colorless oil (88% yield).

No mass peak was observed. The structure of the expected product wasconfirmed via a test reaction: benzylamine (1 eq.), N-methylmorpholine(1 eq.), pentadecyl-4-bromobutanoate (0.9 eq.) were added to a driedheavy walled pyrex tube containing dimethylformamide (1 mL). Thereaction mixture was exposed to microwave irradiation (200 W) for 10 minat 170° C. After the irradiation, the reaction mixture was allowed tocool through an inbuilt system in the instrument until the temperaturehad fallen below 30° C. The resulting reaction mixture was analyzed byLCMS. Molecular weight calculated: 403.64, molecular weight observed:404.50.

Example 7 Synthesis of 4-Bromo-N-Dodecylbutanamide

The title intermediate was synthesized according to the followinggeneral reaction scheme:

4-bromobutanoyl chloride (1.05 eq.) was added in a round bottom flaskcontaining dichloromethane (200 mL) at about 0° C. fitted with apressure-equalizing addition funnel and stirred under nitrogen.Separately, dodecylamine (1 eq.) and triethylamine (1 eq.) weredissolved in dichloromethane (100 mL) and the mixture was transferred tothe addition funnel. This mixture was added very slowly to a roundbottom flask over a period of about 60 minutes. After the completion ofthe addition, the reaction mixture was stirred for another 15 minutes atabout 0° C. and transferred to a separatory funnel. The organic layerwas washed with saturated sodium bicarbonate (×3), water (×2) and brine(×1). The organic layer was dried over sodium sulfate, filtered andconcentrated to give the desired product, 4-Bromo-N-dodecylbutanamide aswhite solid (88.7% yield). Molecular weight calculated: 333.17,molecular weight observed: 334.31.

Example 8 Synthesis of (a) Dipentadecyl4,4′-(3-(methyl(4-oxo-4-(pentadecyloxy)butyl)amino)propylazanediyl)Dibutanoateand (b) Pentadecyl4-(methyl(3-(4-oxo-4-(pentadecyloxy)butylamino)propyl)amino)Butanoate

The mixture of the two title compounds was synthesized according to thefollowing general reaction scheme:

A dry microwave reaction synthesis vessel containing a small magneticstir bar was charged with N1-methylpropane-1,3-diamine (42 mg, 0.48mmole), pentadecyl-4-bromobutanoate (2.1 eq), N,N-diisopropylethyl amine(2.1 eq.) and dimethylformamide (1.0 mL). The vessel was sealed and thereaction mixture was heated up to 90° C. in a CEM microwave synthesizer.After about 20 minutes, the reaction was stopped and the vessel wasallowed to cool down to about 40° C. The analytical LCMS traces of thereaction crude showed the formation of the desired products. The crudematerial was purified by preparative LCMS.

All the fractions containing the desired products were carefullyselected and pooled. The solvent of each fraction was dried down to halfof the original volume under reduced pressure followed by the additionof 1M HCl in methanol (1.0 mL). Finally, all the solvent was removedunder vacuum affording the hydrochloric acid salts of the titlecompounds (a) and (b) shown on the reaction scheme above as whitesolids.

For compound (a): molecular weight calculated is 976.91, molecularweight observed was 977.91. For compound (b): molecular weightcalculated is 680.64, molecular weight observed was 681.64.

Example 9 Preparation of Lipid Compositions for In Vivo Delivery

Some lipids of the present invention were formulated into lipidcompositions, which also comprise cholesterol, poly(ethylene glycol)(PEG) lipids, buffers and ethanol. Animal origin-free cholesterol waspurchased from Sigma-Aldrich (St Louis, Mo.),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (C16 PEG2000 PE) and1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (C14 PEG2000 PE) were purchased fromAvanti Lipids (Alabaster, Ala.). The RNAi was STEALTH™ RNAi having25-base-pair double-stranded RNA oligonucleotides with stabilizingchemical modifications. STEALTH™ RNAi is commercially available fromInvitrogen (Carlsbad, Calif.).

Dry powder lipids were re-suspended in ethanol and the cationic lipid:cholesterol and a PEG lipid were mixed at a weight ratio of 52:20:28respectively. The lipid ethanolic mixture was mixed very rapidly in 200mM Sodium Acetate pH 5.2 solution at a 1:4 ratio. The final formulation(i.e., the total the lipid of the invention plus PEG lipid pluscholesterol) was sterile filtered on a 0.22 μm filter and has aconcentration of 15.625 mg/ml and size 50-80 nm in 50 mM Sodium acetateand 25% Ethanol (preliposomes). The formulations that were made aredescribed in Table 1.

TABLE 1 Lipid Formulations Compound of the Ethanol, Concentration,Invention*⁾ PEG Lipid Helper Lipid Buffer % mg/ml 49 C16 PEG2000 PECholesterol 50 mM Sodium 25 15.625 Acetate 7 C16 PEG2000 PE Cholesterol50 mM Sodium 25 15.625 Acetate 69 C14 PEG2000 PE Cholesterol 50 mMSodium 25 15.625 Acetate 70 C14 PEG2000 PE Cholesterol 50 mM Sodium 2515.625 Acetate 51 C14 PEG2000 PE Cholesterol 50 mM Sodium 25 15.625Acetate 52 C14 PEG2000 PE Cholesterol 50 mM Sodium 25 15.625 Acetate 54C14 PEG2000 PE Cholesterol 50 mM Sodium 25 15.625 Acetate 56 C16 PEG2000PE Cholesterol 50 mM Sodium 25 15.625 Acetate 57 C16 PEG2000 PECholesterol 50 mM Sodium 25 15.625 Acetate 73 C16 PEG2000 PE Cholesterol50 mM Sodium 25 15.625 Acetate 76 C16 PEG2000 PE Cholesterol 50 mMSodium 25 15.625 Acetate 77 C16 PEG2000 PE Cholesterol 50 mM Sodium 2515.625 Acetate *⁾Numbers refer to the numbers of specific compounds ofthe instant invention shown above

The lipoplex (siRNA preliposome complex) was prepared by mixing equalvolumes of preliposome and STEALTH™ RNAi solution diluted to 1.5 mg/mlin water and 25% ethanol. After mixing, the complex was incubated atabout 50° C. for about 30 minutes. After incubation, the lipoplex wasDialysed for about 2 hours in 1 liter PBS 1x pH7.4 using SPECTRA/POR®FLOAT-A-LYZER® G2 8 KDa. After dialysis the volume was measured andadjusted with PBS to the desired siRNA concentration. The lipoplex wasincubated at about 4° C. until in vivo tail vein injection.

Mice were used at ages from 4 to 6 weeks. RNAi against Factor VII (FVII)mouse gene having the Antisense sequence 5′-AUUUGCACAGAUCAGCUGCUCAUUC-3′and the negative control Medium GC content-RNAi were complexed withpre-liposomes as previously described and tested in vivo. 200 μl oflipoplexes containing FVII or CTRL RNAi in PBS were injected per 20 gmouse, by low pressure tail vein injection at a dose of 10 mg/kg and 2mg/kg (siRNA dose). Thirty six hours after tail vein injection livertissue and serum was collected for mRNA and protein knock down analysisrespectively.

Frozen liver tissue was ground into powder and RNA was extracted usingTRIZOL® PLUS RNA Purification System (Invitrogen). Total RNA (about 750ng) for the first strand synthesis was determined using SUPERSCRIPT® IIIRT kit (Invitrogen) and QPCR analysis was performed using taqman assayusing EXPRESS qPCR Supermix Universal (cat#11785-01K).

Factor VII serum protein level was determined as follows Animals wereanesthetized by intramuscular injection ofKetamine/xylazine/Acepromazine (75/5/1 mg/kg respectively), blood wascollected by retroorbital bleed, and serum was processed to measureFactor VII protein level using a chromogenic assay (Biophen FVII, AniaraCorporation) according to manufacturers' protocols.

FIG. 1 provides a graph summarizing the relative % of Factor VII proteinremaining activity, as measured by chromogenic assay, of the differentlipoplex formulations in liver. Referring to the x axis, the numbersrefer to the cationic lipid compounds tested, as shown in Table 1. Thedata demonstrate that such formulations do possess activity in vivo. Noknockdown was observed when the CTRL negative control was injected inthe liver.

FIG. 2 provides a graph summarizing the relative % of mRNA Factor VII asmeasured by qRT-PCR using 2 Taqman assays. In each pairs of bars shownon FIG. 2, the left-hand bar refers to Taqman 29 assay and the righthand bar refers to Taqman 33 assys., The numbers along the x axis referto the cationic lipid compounds tested, as shown in Table 1. The datademonstrate that such formulations do possess activity in vivo. Noknockdown was observed when the CTRL negative control was injected inthe liver.

Example 10 Preparation of Lipid Compositions

The following set of experiments were performed essentially as describedabove in EXAMPLE 9, with the following exceptions. The dialysis stepperformed after formation of the siRNA/lipoplex was not performed.Additionally, the dry powder weight ratios of the IVF 2.0, 57 NO, and 84NO samples was 37.8:10.4:51.8, and the dry powder weight ratios of 57OPT, 72 OPT and 84 OPT samples was 60:7:33. The formulations that weremade are described in Table 2.

TABLE 2 Lipid Formulations Compound of the Ethanol, Concentration,Invention*⁾ PEG Lipid Helper Lipid Buffer % mg/ml IVF2.0 C16 PEG2000 PECholesterol 139.5 mM 25 15.625 Sodium Acetate 57 NO C16 PEG2000 PECholesterol 139.5 mM 25 15.625 (CB00396) Sodium Acetate 57 OPT C14PEG2000 PE Cholesterol 139.5 mM 25 15.625 (CB00396) Sodium Acetate 72OPT C14 PEG2000 PE Cholesterol 139.5 mM 25 15.625 (CB00401) SodiumAcetate 84 NO C14 PEG2000 PE Cholesterol 139.5 mM 25 15.625 (CB00416)Sodium Acetate 84 OPT C14 PEG2000 PE Cholesterol 139.5 mM 25 15.625(CB00416) Sodium Acetate *⁾Numbers refer to the numbers of specificcompounds of the instant invention shown above

FIG. 3 provides a graph summarizing the relative % of Factor VII mRNA asmeasured by qRT-PCR as described above. The results are normalized andare expressed as a % remaining FVII expression (y axis) as a function ofadministered dosage (mg/kg body weight).

Example 11 Preparation of Lipid Compositions for In Vitro Delivery

Some lipids of the present invention were formulated into lipidcompositions, which also comprise cholesterol or DOPE, buffers andethanol Animal origin-free cholesterol was purchased from Sigma-Aldrich(St Louis, Mo.) and DOPE was purchased from Avanti Lipids (Alabaster,Ala.). Silencer® Select CSNK2A1 siRNA and Silencer® Select negativecontrol siRNA (cat #4390824 siRNA id# s3637 and cat #4390843) having theantisense sequence AAACUAUAAUCGUACAUCUGA and UUACGUCGUCGCGUCGUUATT,respectively, were resuspended with nuclease-free water to a stockconcentration of 50 μM, which were further diluted to meet downstreamexperiments and is commercially available from Life Technologies(Carlsbad, Calif.).

For compounds 83 and 67, the dry powder lipids were re-suspended inethanol at a final concentration of 1 mg/ml and either used as lipidalone (Compound 83) or combined with cholesterol in 1:0.5 ratio(Compound 67). The formulations tested were made as described in Table 3

TABLE 3 Lipid Formulations Compound of the Helper Ethanol, Invention*Lipid % 83 None 100 67 Cholesterol 100 *Numbers refer to the numbers ofspecific compounds of the instant invention shown above

Primary rat cortex neuronal cells were purchased from GIBCO (Cat. No.A10840) and maintained in Neurobasal™ medium (cat #21103-049)supplemented with 2% B-27® Serum-free (50x cat #17504-044) and 0.5 mMGlutaMAX™ (cat #35050-061). Primary rat hippocampal neuronal cells werepurchased from GIBCO (Cat. No. A10841) and maintained in Neurobasal™medium (cat #21103-049) supplemented with 2% B-27® Serum-free (50x cat#17504-044), 0.5 mM GlutaMAX™ (cat #35050-061) and 25 μM L-Glutamate(Sigma cat # G-2834). Hela cells were purchased from ATCC (ATCC # CCL-2)and maintained DMEM, high glucose, GlutaMAX™, pyruvate (cat #10569-010)supplemented with 10% fetal bovine serum, US certified, heat inactivated(cat #10082-139) without antibiotics

Neuronal cells were plated in 96-well plates coated poly-d-lysine twodays before transfection at 10K cells/well. Hela cells were plated in96-well plates one day prior to transfection at 25K/well. siRNAs (3pmol) were diluted 20 ul nuclease-free water at a final concentration of30 nM in 1.5 mL tubes. Master mixes were made to cover all wells to betransfected for each siRNA. Lipid dilutions were prepared by pipetting0.15, 0.3 and/or 0.6 μl/well or 0.3 ul LIPOFECTAMINE™ RNAiMAX as acontrol in Opti-MEM® I in total final volume of 10 μl/well. The lipidand siRNA complexes were prepared by adding 10 ul of the lipid mixtureto 20 μl of diluted siRNA. The complexes were mixed by pipetting up anddown and incubated at room temperature for 10 minutes. After the 10minute incubation, 30 μl of the complex was then added to the pre-platedcells containing 120 ul of growth medium cultured in 96-well platescontaining 150 ul. After 24 hours, cells were harvested with the TaqMan®Gene Expression Cells-to-CT™ kit (cat #AM1728, Life Technologies)according to the manufacturer's protocol. After cells were lysed,reverse transcription (RT) reactions were set-up followed by real-timePCR using TaqMan Gene Expression assays for the target gene of interest(CSNK2A1 (Hs00953536_m1 for human or Rn00587582_m1 for rat)) andeukaryotic18s rRNA (4333760T) was used as the endogenous control.

FIG. 4 depicts a graph summarizing the relative percent of CSNK2A1remaining activity normalized to negative control as measured by qPCRassay of the Compound 83 lipoplex formulations in HeLa cells, ratprimary cortical neurons and rat primary hippocampal neurons. Referringto the x axis, the numbers refer to the final volume of cationic lipidformulation summarized in Table 3 for each well.

FIG. 5 provides a graph summarizing the relative percent of CSNK2A1remaining activity normalized to negative control, as measured by qPCRassay of Compound 67 lipoplex formulations in Hela, primary corticalneurons and primary hippocampal neurons. Referring to the x axis, thenumbers refer to the final volume of cationic lipid per well tested, asshown in Table 3. The data demonstrate that such formulations do possessactivity in vitro.

The above results demonstrate that the formulation s described above areefficient at introducing siRNA molecules to various organs in animals invivo as well as to animal and human cells cultured in vitro.

Example 12 Delivery of mRNA and Expression In Vivo Preparation ofTransfection Lipids

Cationic lipid 87 (shown above) was synthesized at Life Technologies,Carlsbad, Calif. according to methods described herein; Animalorigin-free Cholesterol was purchased from Sigma-Aldrich (StLouis),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (C16PEG)) was purchased from Avanti LipidsAlabaster. CB547, Cholesterol and C16PEG were diluted in 100% ethanol at40° C. The lipid mixture was then mixed in 200 mM Sodium Acetate, pH 5.2using a syringe equipped with a 27G needle at a flow rate of 20ml/minute. The formulation was then stored at 4° C.

Preparation of CRE mRNA

The cDNA sequence for CRE that was used in the experiments set forthbelow was as follow:

TTGGACCCTCGTACAGAAGCTAATACGACTCACTATATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGCAACTTTTCTATACAAAGTTGCTATGGGCCCAAAGAAGAAGAGAAAGGTTTCGAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAGACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCATGCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGAT

A DNA plasmid containing the cDNA sequence was synthesis (GENEART® GeneSynthesis) and cloned into a plasmid DNA vector.

Purified plasmid DNA is digested with Ase I restriction enzyme (NewEngland Biosciences, Cat. No. R0526) according to manufacturer'sprotocol. The linearized DNA was purified using PureLink™ PCRpurification kit (Life Technologies, Cat. No. K310001) according tomanufacturer's protocol, and eluted with purified water. DNAconcentration was determined by UV absorbance. The Promega RiboMAX™Large Scale RNA production System-T7 (Cat. No. P1300) was utilized tosynthesize mRNA according to manufacture's protocol. In each reaction,5-10 μg of linearized DNA yielded 200-250 μg of mRNA. Followingsynthesis, mRNA was purified using phenol:chloroform extraction followedby ethanolic precipitation. The mRNA product was resuspended in purifiedwater and the concentration was determined. The mRNA was cappedutilizing ScriptCap™ m⁷G Capping System (Cat. No. C-SCCE0625) andScriptCap™ 2′-O-Methyltransferase Kit (Cat. No. C-SCMT0625), both fromCellScript™. The capped mRNA was polyadenylated using Epicentre®Poly(A)Polymerase Tailing Kit (Cat. No. PAP5104H). The final product ispurified again via phenol:chloroform extraction followed by nucleic acidprecipitation. The purified mRNA is resuspended in purified water andconcentration determined. Concentration was adjusted to 3 mg/ml in waterand stored at −80° C.

Preparation of Transfection Complexes Containing Lipid 87 and Cre mRNA

Concentration of mRNA synthesized in the previous step was adjusted to0.3 mg/ml in water containing 25% ethanol. The lipoplex (mRNApreliposome complex) was prepared by mixing equal volumes of preliposomeand mRNA solution. After mixing, the complex was incubated at 50° C. for30 minutes, then dialysed for 2 hours in 1 liter phosphate bufferedsaline (PBS), pH 7.4 using Spectra/Por® Float-A-Lyzer® G2 8 kDa. Afterdialysis, the volume was measured and adjusted with PBS to the desiredmRNA concentration. The lipoplex was stored at 4° C. until in vivoinjection.

Intravenous Injection of In Vivo Fectamine-547 mRNA Complex.

All procedures used in animal studies were approved by the InstitutionalAnimal Care and Use Committee (IACUC) and were consistent with local,state and federal regulations as applicable Mice were purchased fromJackson laboratories (B6.129S4-Gt(ROSA)26Sor^(tm3(CAG-luc)Tyj)/J) Thismouse bears a CMV driven luciferase reporter gene with a loxP-flankedSTOP codon under the control of a CMV promoter. In the presence of Crerecombinase, the loxP sites recombine to excise the STOP codon, therebyallowing translation of the luciferase reporter protein.

For the lacZ experiments, mice aged from 4 to 6 weeks carrying lacZ gene(B6.129S4-Gt(ROSA)26Sor^(tm1Sor)/J) were purchased. Expression of lacZwas determined by qRT-PCR using standard methods. Two 200 μl of lipoplexcontaining mRNA CRE in PBS prepared as described above were injected permouse by low pressure tail vein injection at a dose of 1.5 mg/kg and 0.5mg/kg (mRNA dose). For luminescence imaging, mice received 150 mg offirefly luciferase (Biosynth AG, Staad, Switzerland) per kg body weightgiven i.p. After anesthesia with isoflurane gas (Abbott Laboratories,North Chicago, Ill.), the mice were placed into a Xenogen IVIS imagingstation (Xenogen Corp., Alameda, Calif.) and imaged using Living ImageSoftware (Xenogen Corporation).

FIG. 6A shows whole body images of mice treated with a transfectioncomplex containing Cre mRNA and lipid 87 at 1.5 mg/kg (left), 0.5 mg/kg(middle) or PBS (right). Luciferase expression was measured usingXenogen IVIS and signal was quantified.

FIG. 6B shows whole mount lung, heart, spleen and liver (as indicated)organs dissected from each of the two treated mice shown in FIG. 6A.Luciferase expression measured on Xenogen IVIS. Luciferase expressionwas detected in Liver and Spleen. No luciferase expression was detectedin PBS injected mice (FIG. 1).

FIG. 7 A-F is a graphical representation showing analysis of LacZ geneexpression as measured by qRT-PCR. Mice treated with a transfectioncomplex containing Cre mRNA and lipid 87 displayed a much strongerexpression of the lacZ gene in Spleen (compare spleen control HMBSexpression FIG. 7A with spleen LacZ expression FIG. 7B), Liver (compareliver control HMBS expression FIG. 7C with liver LacZ expression FIG.7D) compared to control as determined by Cts. cDNA input was normalizedusing HMBS. No increase expression of lacZ was observed in Kidney (FIG.7E and FIG. 7F).

Although only a few embodiments have been described in detail andexemplified above, those having ordinary skill in the art will clearlyunderstand that many modifications are possible in the describedembodiments without departing from the teachings thereof. All suchmodifications are intended to be encompassed within the followingclaims.

1-22. (canceled)
 23. A transfection complex comprising anamine-containing transfection reagent having the general structure I, orpharmaceutically acceptable salts thereof:

wherein: each of X₁ and X₂ is a moiety independently selected from thegroup consisting of OS, N-A and C-A, wherein A is selected from thegroup consisting of hydrogen and a C₁-C₂₀ hydrocarbon chain; each of Yand Z is a moiety independently selected from the group consisting ofC═O, C═S, S═O, CH—OH and SO₂; each of R₁, R₂ and R₃, R₄, R₅, R₆ and R₇is a moiety independently selected from the group consisting ofhydrogen, a cyclic or an acyclic, substituted or unsubstituted, branchedor unbranched aliphatic group, a cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic group, asubstituted or unsubstituted, branched or unbranched acyl group, asubstituted or unsubstituted, branched or unbranched aryl group, asubstituted or unsubstituted, branched or unbranched heteroaryl group; xis an integer independently having the value between 1 and 10,inclusively; n is an integer independently having the value between 1and 3, inclusively, m is an integer independently having the valuebetween 0 and 20, inclusively, p is an integer independently having thevalue of 0 or 1, wherein if m=p=0, then R₂ is hydrogen, with the furtherproviso that if at least one of n or m has the value of 2, then R₃ andnitrogen in structure I form a moiety selected from the group consistingof:

wherein each of g, e and f is an integer independently having the valuebetween 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, andeach * indicates the nitrogen atom in structure I.
 24. The transfectioncomplex of claim 23, or pharmaceutically acceptable salts thereof,wherein each of R₁ and R₂ is independently selected from the groupconsisting of substituted or unsubstituted, branched or unbranched alkylor alkenyl groups having between 3 and about 20 carbon atoms, andbetween 0 and 4 double bonds.
 25. The transfection complex of claim 24,or pharmaceutically acceptable salts thereof, wherein each of R₁ and R₂is independently selected from the group consisting of substituted orunsubstituted, unbranched alkyl or alkenyl groups having between 8 andabout 18 carbon atoms, and between 0 and 2 double bonds.
 26. Thetransfection complex of claim 23, or pharmaceutically acceptable saltsthereof, wherein each of n and m independently has the value of 1 or 3,and R₃ is selected from the group consisting of

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structure I,wherein each H on any * position can be replaced to achieve theattachment to the nitrogen atom in structure I.
 27. The transfectioncomplex of claim 23, or pharmaceutically acceptable salts thereof,wherein x has the value between 1 and
 6. 28. The transfection complex ofclaim 23, wherein the amine-containing transfection reagent has thegeneral structure II, or pharmaceutically acceptable salts thereof:


29. The transfection complex of claim 28, or pharmaceutically acceptablesalts thereof, wherein at east one X₁ is NH.
 30. The transfectioncomplex of claim 28, or pharmaceutically acceptable salts thereof,wherein at least one X₁ is O.
 31. The transfection complex of claim 28,or pharmaceutically acceptable salts thereof, wherein each R₁ isindependently selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl or alkenyl groups havingbetween 3 and about 20 carbon atoms, and between 0 and 4 double bonds.32. The transfection complex of claim 31, or pharmaceutically acceptablesalts thereof, wherein each of R₁ is independently selected from thegroup consisting of substituted or unsubstituted, unbranched alkyl oralkenyl groups having between 8 and about 18 carbon atoms, and between 0and 2 double bonds.
 33. The transfection complex of claim 28, orpharmaceutically acceptable salts thereof, wherein each of n and mindependently has the value of 1 or 3, and R₃ is selected from the groupconsisting of

wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows apotential point of attachment of R₃ to the nitrogen atom in structureII, wherein each H on any * position can be replaced to achieve theattachment to the nitrogen atom in structure II.
 34. The transfectioncomplex of claim 28, or pharmaceutically acceptable salts thereof,wherein x has the value between 1 and
 6. 35. The transfection complex ofclaim 23, or pharmaceutically acceptable salts thereof, the compoundbeing selected from the group consisting of compounds 1-87 or isomersthereof:


36. The transfection complex of claim 23, further comprising at leastone helper lipid.
 37. The transfection complex of claim 36, wherein thehelper lipid is selected from the list consisting of BMOP(N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimunbromide), DDPES (Dipalmitoylphosphatidylethanolamine5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation ofcetyltrimethylammonium bromide (CATB) and DOPE), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE(dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine),DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC(Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine),DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC(dioleoydimethylammonium chloride), DOSPER(1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA(N-[1-(2,3-dioleyloxyl)propyl]-n,n,n-trimethylammoniumchloride), DDAB(didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate),DOTAP.Cl, DC-chol(3,β-N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA(2-(sperminecarboxamido)ethyl)-N,N-dimethy-lammonium trifluoroacetate),DC-6-14 (O,O′-Ditetradecanoyl-N-(alphatrimethylammonioacetyl)diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE(dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP(1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA(2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminiumtrifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE(N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME(N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammoniumiodide), GAP-DLRIE:DOPE(N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide/dioleyl phosphatidylethanolamine), DPPC(Dipalmitoylphosphatidylcholine), DOPG(1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl),N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivativesthereof); phosphotidylethanolamine (and derivatives thereof);phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPEdipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, O-Chol (3beta[1-ornithinamidecarbamoyl] cholesterol), POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; piperazine-based cationic lipids,phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG(dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;phosphatidylserine (and derivatives thereof); phosphatidylserines, suchas dioleoyl- or dipalmitoylphosphatidylserine; diquaternary ammoniumsalts such as N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,2-ethanediamine(TmedEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,3-propanediamine(PropEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,6-hexanediamine(HexEce), and their corresponding N,N′-dicetyl saturated analogues(TmedAce, PropAce and HexAce), diphosphatidylglycerols; fatty acidesters; monocationic transfection lipids such as1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol;1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol;1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol;1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βOH-sterolsas well as derivatives thereof phosphatidyl choline or commerciallyavailable cationic lipid mixtures such as, for example, LIPOFECTIN®CELLFECTIN® (1:1.5 (M/M) formulation ofN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitylspermine (TMTPS)and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXN™,TRANSFAST™, TRANSFECTAM®, TRANSMESSENGER®, vectamidine(3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine(diC14-amidine), OLIGOFECTAMINE®.
 38. The transfection complex of claim23, further comprising at least one pegylated lipid.
 39. Thetransfection complex of claim 23, further comprising at least onebioactive agent.
 40. The transfection complex of claim 39, wherein thebioactive agent is a DNA molecule, and RNA molecule, a protein of adrug.
 41. The transfection complex of claim 40, wherein the RNA moleculeis an siRNA, an shRNA, an miRNA and stRNA or an mRNA.
 42. Thetransfection complex of claim 39, wherein the bioactive agent is ansiRNA molecule.
 43. The transfection complex of claim 39, wherein thebioactive agent is an mRNA molecule.
 44. The transfection complex ofclaim 39, wherein the bioactive agent is an DNA molecule.
 45. Thetransfection complex of claim 23, further comprising a targeting moiety.46. The transfection complex of claim 23, further comprising a targetingmoiety selected from the list consisting of peptide, a modified peptide,an antibody, a modified antibody, a receptor molecule, a modifiedreceptor molecule, a single or a double stranded nucleic acid molecule,a modified single or double stranded nucleic acid molecule, a peptide ornucleic acid aptamer, a modified peptide or nucleic acid aptamer, anorganic molecule, a polysaccharide.